Abstract of thesis entitled
Transcripción
Abstract of thesis entitled
Title The localization of E. coli persistent gene products Advisor(s) Huang, J; Zhou, Z Author(s) Miao, Yuanying.; 缪元颖. Citation Issued Date URL Rights Miao, Y. [缪元颖]. (2010). The localization of E. coli persistent gene products. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4555479. 2010 http://hdl.handle.net/10722/143999 The author retains all proprietary rights, (such as patent rights) and the right to use in future works. Abstract of thesis entitled The Localization of E. coli Persistent Gene Products Submitted by Miao, Yuanying for the Degree of Doctor of Philosophy at the University of Hong Kong in December 2010 Despite the genomes of many organisms being completely sequenced, the functions of a large proportion of their genes still remain unknown. Having knowledge of the subcellular localization of proteins should provide insights into their functions. Visualization of fluorescent protein labeled proteins in live cells using fluorescence microscope became an effective method to determine the localization of proteins. Recombineering technology allows the efficient integration of transformed linear double stranded DNA, which contains short homologous regions (35-55 bp), into the chromosome. In order to produce the EGFP fusion proteins under the control of the endogenous promoter of the target gene, double stranded DNA targeting cassettes generated by PCR is transformed into the E. coli strain that contains the recombineering system by electroporation. This PCR product contains the coding region of EGFP and a selectable marker and 50 bp homologous sequences to the chromosome regions immediately upstream and downstream of the insertion site. The correct EGFP-tagged strains are selected by selective medium and confirmed by colony PCR. The localization pattern of target gene product is determined by observing the fluorescence signal under fluorescence microscope. All 611 persistent genes of E. coli have been tried to tag and localize. Among the 611 genes, 189 genes have been tagged successfully and 139 gene products have been located successfully. After overnight cell growth, 115 of these proteins were diffusely located in the cell, 12 were located at foci at one or both poles or the center of the cell, and 8 were located at the membrane, 4 proteins localized diffusely throughout the cytoplasm as well as in 1–4 foci.Several proteins show different localization pattern from previous research results. Analysis of the dynamic distributions of the tagged proteins provided fresh insights into the functions of the persistent genes. EGFP-tagged strains show different fluorescence intensity under different growth conditions, suggesting that the tagged strains may be a potential resource for the study of gene expression level. Several proteins were identified to have different subcellular locations during different growth phases, suggesting that the growth phase of strain should be considered when the localization patterns of proteins are determined. Other researchers may access the data through the E. coli persistent proteins localization database we established. (http://www.biochem.hku.hk/huanglab/Colilo/index.php) (Words: 367) The Localization of E. coli Persistent Gene Products by Miao, Yuanying (缪元颖) B.Sc. Sichuan University M.Sc. Chengdu Institute of Biology, Chinese Academy of Science A thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy at The University of Hong Kong December 2010 Declaration I declare that the thesis and the research work there of represents my own work, except where due acknowledgement is made, and that it has not been previously included in a thesis, dissertation or report submitted to this University or to any other institution for a degree, diploma or other qualifications. Signed: ………………………………………………… Miao, Yuanying i Acknowledgements I would like to express my greatest gratitude to my supervisor, Dr. Jiandong Huang, for giving me an invaluable opportunity to study in his laboratory and for his patience and guidance. I will never forget those informative and useful discussions with him; I will never forget his authentically encouragement when I feel puzzle and depression. I would like to thank my co-supervisor, Dr. Zhongjun Zhou, for his guidance and helpful advice. Special thanks Professor Antoine Danchin for his invaluable advice to my project. I also want to thank Dr. Rory M. Watt and Dr. Jing Wang for their selfless help when I start my PhD project. I am also grateful to Dr. Ye Jin and Ms. Suisui Dong for their selfless help and discussion. Many thanks to all my labmates, Dr. Yanxiang Ni, Dr. Guixia Zhu, Dr. Zai Wang, Dr. Linyu Lu, Dr. Shing-Yan Huen Michael, Mr. Song Lu, Ms. Shuang Qi, Ms. Mei Yang, Mr. Chenli Liu, Ms. Huiyan Gan, Ms. Ju Cui, Mr. Bin Yu, Mr. Raozhou Lin, Ms. Lei Shi, Ms. Songyue Zheng, and other friends for their help. I would like to thank Mr. Hari Krishna Yalamanchili for helping me solve problems when I upload data to our database. I am also thanksful to my friend, Mr. Kai Huang, thank you for your help and discussion. I would also like to thank Ms. Chan Ling Chim, Jess and other technicians for their technical support and all of the administrative staff members of Department of Biochemistry for their help. ii I thank my friend, Dr. Nina L Cheung, for helping me with my English writing. Thanks to The University of Hong Kong for offering me a postgraduate studentship (2006-2010) and thanks to CRCG for offering me a conference grant. I would like to thank my parents and my sister. I cannot complete my PhD study without their support and encouragement. Your love will encourage me to achieve my goal in life. Last but not least, I thank my wife for her support and tolerance. I apologize for not being stay with you in these years. iii Table of Content Declaration……………………………………………………………i Acknowledgements…………………………………………………..ii Table of Content……………………………………………………..iv List of Figures……………………………………………………...viii List of Tables………………………………………………………...x 1 Chapter 1: Introduction 1.1 Protein localization and function 1 1.1.1 Protein subcelluar localization 1 1.1.2 Methods to determine protein subcellular 3 localization 1.1.3 Protein subcelluar localization in eukaryote 6 1.1.4 Protein subcelluar localization in prokaryote 8 1.2. Essential genes and persistent genes 11 1.3.Homologus recombination and recombineering 13 1.3.1 Homologous recombination 13 1.3.1.1 Homologous recombination in E. coli 13 1.3.1.2 Homologous recombination in eukaryotes 16 18 1.3.2 Recombineering 1.3.2.1 Concept and mechanism of recombineering iv 18 1.3.2.2 Application of recombineering 20 1.4 Green Fluorescent Protein (GFP) 21 1.4.1 Properties of GFP and its variants 21 1.4.2 The application of GFP in prokaryotes 24 1.4.3 The application of GFP in eukaryotes 27 28 1.5 Aims of the Project Chapter 2: Materials and Methods 30 30 2.1 Materials 2.1.1 Bacterial strains 30 2.1.2 Plasmid and primers 30 2.1.3 Agarose, Antibiotic and Culture Medium 32 32 2.2 Experiment methods 2.2.1 Preparation of component cells for 32 plasmid amplification and plasmid transformation 2.2.2 Plasmid DNA purification 33 2.2.3 Amplification of linear dsDNA targeting 33 cassettes by Polymerase Chain Reaction (PCR) 2.2.4 PCR Product Purification 34 2.2.5 Restriction Enzymes Digestion of plasmids 34 of purified PCR product 2.2.6 λ-Red mediated dsDNA recombination 35 protocol 2.2.7 PCR screening of recombinant clones v 35 36 2.2.8 Fluorescence microscopy 2.2.8.1 Cell culture condition 36 2.2.8.2 Staining chromosome with DAPI 36 36 2.2.9 Time–lapse experiment 38 Chapter 3: Results 3.1 Construction of an EGFP-tagged library of 38 persistent genes 3.1.1 Preparation of the tagging cassette by PCR 38 3.1.2 Tagging CDS of persistent genes in 40 chromosomal and positive clones selection 3.2 Subcellular localization of E. coli persistent 43 proteins and subcellular localization of ribosomal subunit proteins of E. coli 3.3 Cell cycle and growth phase-specific protein 57 localization pattern 3.4 ColiLo: an on-line catalogue of protein 60 localization database for E. coli persistent proteins Chapter 4: Discussion and Perspective 62 4.1 Organization of the proteome 62 4.2 Relationship of protein localization and function 64 4.3 The dynamic of protein localization 68 4.4 Conclusions and perspective 70 vi 73 References Appendix 1 The fluorescence images of successfully tagged E. coli persistent genes 99 Appendix 2 The list of all 611 persistent genes of 135 E. coli Appendix 3 Primers used in the study of protein localization of E. coli vii 163 List of Figures Figure 1 Map of plasmid used as PCR template for producing the tagging cassette ………………………………………31 Figure 2 Purified PCR product of targeting cassette......... 39 Figure 3 The strategy used to create chromosomal C-terminal fluorescent fusion proteins using a combination of λ - Red mediated homologous recombination and Cre/LoxP mediated site-specific recombination, as exemplified by the construction of suhB-EGFP……………………………….41 Figure 4 PCR screening of correct recombinant spoT EGFP tagged clones………………………………………………42 Figure 5 Typical fluorescence microscopy images of live, immobilized E. coli DY330 cells containing a 3' EGFP coding sequence fused to a persistent gene on the chromosome……………………………………………….54 Figure 6 (A) Time-lapse microscope image of E. coli with EGFP-tagged rpsO (B) A schematic diagram of the change of the RpsO protein localization pattern during cell division …………………………………………………………….56 viii Figure 7 Change of localization pattern at different growth phases……………………………………………………...59 Figure 8 The homepage and search result pages (GroS protein localization page as an example) of ColiLo database …….61 Figure 9 The localization pattern of the EGFP tagged recA gene product……………………………………………….64 Figure 10 The localization pattern of RpsO-EGFP at different growth temperatures……………………………………….67 ix List of Tables Table 1 There different protein tagging locations……..........5 Table 2 Experimental results of gene tagging and protein localization ………………………………………………...45 Table 3 E. coli fluorescent protein localization……………45 Table 4 List of localization pattern of the succeed localized proteins…………………………………………………….46 Table 5 Localization patterns of EGFP-tagged ribosomal subunit proteins (rsp)………………………………...........55 x Chapter 1: Introduction 1.1 Protein localization and function Following the development of genome sequence technology, many whole genome sequencing projects have been completed. One of the important results of the genome sequencing projects is that many new proteins have been discovered. The next and the most important step, is to discover the functions of their genes and the functional protein networks within the cell. In order to perform their function, proteins must be located to the appropriate compartment in the cell. In general, proteins can be found in four compartments of E.coli: cytopalsm, periplasm, inner membrane and outer membrane. In cytoplasm, two major types of localization patterns are found: diffusion or form foci. The typical example of relationship between protein subcelluar localization and its function is the dynamic localization of FtsZ, one of the proteins involved in cell division. It is found that FtsZ is diffusely located in the cell before cell division. However, it localizes to the mid-point of the cell and forms a ring (FtsZ-ring) as a scaffold for the recruitment of other cell division protein, after the cell begins to divide (1). Since a protein‟s function is closely related to its subcellular localization, studying protein localization should provide invaluable information about the protein‟s function. 1.1.1. Protein subcelluar localization Compartmentalization is very important to maintain normal physiologic function of a cell because it provides different microenvironments that makes enzymes and their substrates physical proximity and thus beneficial to the compartment special metabolic reactions. Due to different metabolic activities are carried out in different compartment, the proteins in each corresponding cell compartment are involved with their own unique metabolic activities. Unlike eukaryotic cells that are subdivided into functionally compartments, such as cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic 1 reticulum (ER), etc, bacterial cells lack nucleus and membrane-bound organelles. However, protein localization in prokaryotes is still an important aspect for the normal functions of a protein. One of the methods in studying protein functions is to identify their subcellular localization since the functioning of cellular processes and pathways requires individual proteins to be at defined sites at the correct time. In bacterial cells, following synthesis in cytoplasm, the proteins stay in cytoplasm or are directed to the target location with several different transport systems. These transport systems include Type I secretion system, which transports new synthesized proteins directly from the cytoplasm to the extracellular space; Type II secretion system, which inserts proteins into or translocation across the cytoplasmic membrane; Type III secretion system, which involves injecting virulence proteins of pathogenic bacteria into host cells directly; Type IV secretion system, which transport bacterial protein or DNA effector molecules into a eukaryotic cell directly; Type V secretion system, which involves self-translocating across the outer membrane of Gram-negative bacteria (2). In eukaryote, following synthesis in cytoplasm, secretory proteins have special sequence in their precursor protein which transports into Endoplasmatic Reticulum (ER) and then move to other target compartments or stay in the ER. Other proteins that are produced on free cytoplasmic ribosomes are transported to the target organelle under the direction of the N-terminal signal sequences which will be cleaved by special peptidases. It is essential to its function that the correct protein is transported to the targeted organelles (3). For example, membrane proteins are a series of proteins that are attached to the cell membrane and play important roles in immune response and transportation. In order to locate them on the membrane, a signal peptide that lead protein to the membrane can be found at the N-terminal portion of these membrane proteins. The close relationship between protein localization and its functions shows the important of studying protein functions. 2 1.1.2 Methods to determine protein subcellular localization There are two major research methods on determining protein localization: experimental and computational. The experimental methods include biochemical separation method and tagging method. The former method is purification of different compartments (or organelles) with biochemical technology, like density gradient centrifugation and then analysis the proteins in this compartments (or organelles) by various technological means, such as two dimensional gel electrophoresis and MS, LC-MS (4-6). The advantage of this method is that it doesn‟t interfere with the normal expression of the proteins and give the complete protein localization information. But the biggest challenge of biochemical separation method is the production of pure cell organelles without contamination from other parts of the cell because the contaminating proteins will affect the accuracy of the localization results (4, 5). The tag method involves labeling target proteins with short peptides (e.g. epitope) or proteins (e.g. Green Fluorescent Protein, GFP) at the N- or Cterminus. These peptides or proteins can be detected by biochemistry (e.g. Western blot analysis) or biophysical methods (e.g. GFP by fluorescence). Many epitope tags have been developed to label proteins for various purposes, like protein purification and protein localization. The most used epitope tags include V5-tag, c-myc-tag, HA-tag, and etc. The disadvantage of using epitope tags is the complex and time-consuming process of detecting the epitope. With development of various fluorescent proteins, determing localization of proteins by using fluorescent proteins as tags has become the main method. The advantages of fluorescent proteins, for example, no need for an additional substrate for its expression, and easy detection by appropriate UV illumination, made it popular in the study of protein localization. Agaton et al reported another protein localization method in a mammalian cell. The monospecific polyclonal antibodies are generated by cloning and expressing selective proteins fragments (PrESTs) against corresponding proteins. These antibodies are successfully used to generate protein profiles and protein localization data by immunohistochemistry (7). On the basis of this method, Uhlen et al construct a protein atlas for expression and localization profiles 3 with 700 antibodies against human proteins from normal and cancer tissues (8). In tagging method, the tags can be inserted into different locations of target proteins: internal, N-terminus and C-terminus. The differences of three tagging locations are shown in Table 1 (9-14). The C-terminal tagging method is the easiest and it is also the method that causes least disruption to the function of target proteins among these three methods. In my study, I choose to tag the proteins at C-terminus. 4 Table 1 Three different protein tagging locations Tagging location Schematic diagram Tagging method Disadvantages Easy to disrupt the function of target Internal Transposon proteins The introduction of a tag between the promoter and the coding sequence may lead to alteration in gene expression Homologus N-terminal Recombination level and change the subcellular localization; disturb the protein localization due to interference with the signal sequence; sometimes disrupt protein function. Homologus C-terminal Recombination 5 Sometimes disrupt the target protein function The computational method is the new localization technique which developed recently through the generation of massive amount of sequence data. Any one or combination of overall protein amino acid composition, known target sequences, sequence homology, sequence motifs can become the basis of computational methods (2). Computational methods have been widely used in predicting protein localization of both eukaryotes and prokaryotes. PSORT, which predict protein localization based on overall protein amino acid composition and the N-terminal targeting sequence and motifs, was the first localization prediction method developed for bacteria (2). Other typical computational methods include: TargetP, the most comprehensive method based on N-terminal targeting sequence (3, 15) and Cello, which can predict five subcelluar localizations in Gram-negative bacteria (3, 16), for example. The disadvantages of computational protein subcellular localization predict methods are: the limitations on the number of predicted location; number of proteins which contain signal peptide sequences or with prior structural/functional information; and the inability to represent the entire proteomes (17). This thesis will use the experimental method in obtaining more information about the protein subcellular localization. 1.1.3 Protein subcelluar localization in eukaryote In eukaryotic cells, compartmentalization is an important approach to achieve restricted protein localization. Membrane-bounded organelles such as the nucleus, mitochondria, Golgi complex, and endoplasmic reticulum are key compartments to restrict protein localization. As one of the model organisms, many research works on yeast protein localization have been carried out by using various tagging methods. By using immunofluorescence, Burns et al identified 245 lacZ-fusion Saccharomyces cerevisiae proteins with rabbit anti-β-gal antibody and donkey anti-rat antibodies linked to fluoresceinisothiocyanate (18). Similarily, another research team labeled the genome of Saccharomyces cerevisiae with HAT tag using transposon-tagging strategy and 1,340 diploid strains 6 were examined by indirect immunofluorescence with antibodies directed against the HA-epitope. Of these examined strains, 415 are located successfully and tagged proteins can be found at various compartments, i.e. mitochondria, nucleolus, plasma membrane, etc (19). In order to characterize the subcellular localization of more yeast proteins, Kumar et al employed high-throughput methods of epitope-tagging and immunofluorescence analysis and determined the localization of more than 3300 proteins, about 55% of the yeast proteome (20). Determining the protein localization by labeling target proteins with proteins, like lacZ HAT, is not a direct localization method because it needs to detect tagging proteins with another protein. However, with the observation of Green Fluorescent Protein (GFP) under fluorescence microscopy, the localization of labeled proteins can be determined directly, making it easier and less costly. Niedenthal et al constructed N-terminal GFP-fusion expression vector (pGFP-N-FUS) and C-terminal GFP-fusion expression vector (pGFP-C-NUS) and transformed the fused pGFP-N-FUS into yeast cells after the insertion of three genes into the pGFP-N-FUS vector. The localization of these three N-labeled proteins is determined by observing fluorescence signal under fluorescence microscopy. Although it cannot exclude the possibility of GFP-fusion proteins causing an error, the researchers concluded GFP as an invaluable tool in analyzing protein localization pattern in yeasts via the three tagged proteins mentioned in the above experiment (21). Unlike labeling specified protein, a 3‟ end GFP-fusion genomic DNA library of yeast Schizosaccharomyces pombe is established by Ding et al, plasmids isolated from 516 transformants shows distinct fluorescence localization patterns, and 250 independent genes are determined by DNA sequencing. Some new intracellular structural components were found in this work and the dynamics of a GFP labeled protein was also observed by a time-lapse experiment. The most important section from the research are the factors authors mentioned that may influence the correct of localization results by analysis of the GFP-fusion genomic DNA library: First, the bias exists due to the sequenced 512 clones only containing 250 genes while other genes have 7 not been cloned due to several reasons as in the fusion of GFP disturbing the localization of the labeled proteins and that the C-terminal tagging truncates the localization signal, for example. Secondly, the linker between GFP and the target gene also affect the protein localization. The authors conclude that tagging GFP directly to the target protein or with a linker made up of heterocyclic aromatic amino acids may destroy the intracellular localization of target proteins. These factors all should be considered when determining protein localization of proteins using the labeling method (22). Similarly, another research team determined the localization of 4431 proteins, which is about 90% of the Schizosaccharomyces pombe proteome by integrating pDUAL-YFH1c vector that contain ORF-YFP into the chromosome (23). GFP tagged method also can be used in the study of proteins localization of other yeast species. Huh et al inserted PCR cassettes containing GFP directly onto the C-terminus of a targeted gene on the chromosome of the budding yeast Saccharomyces cerevisiae and identified subcellular localization of 4156 proteins, which represents about 75% of yeast genome, of which 70% were not localized previously. Importantly, they found that co-localization strongly correlates with biological function. These research results further confirmed that the protein localization can be one of the effective methods in determining protein function (24). 1.1.4 Protein subcelluar localization in prokaryote Although prokaryotic cells do not have such complex structures as in eukaryotes, their cellular spaces are also highly organized. Identifying all the proteins from different subcellular structures is a key step towards a comprehensive understanding of cellular biology. In the study of prokaryotes, as the model organism, most research work has focused on E. coli. But little research has been on global proteins‟ subcelluar localization; with more works concentrating on the subcellular localization of some special proteins. For example, ftsk gene is essential to the division of E. coli. By tagging FtsK with GFP, Yu et al determined that FtsK is located on the septum and also found 8 that the N-terminal region determines the correct location of FtsK. A single amino acid mutation at the N-terminal region can inhibit the FtsK location at the septum (25). Up til now, several approaches have been reported on the localization of E. coli proteins on a larger scale. Ellison and colleagues biochemically fractionated E. coli cells into cytosol, periplasm, inner membrane, and outer membrane components. They then determined protein identity, location, abundance, modification state, apparent isoelectric points, and molecular mass using high resolution two-dimensional electrophoresis protein gels and tandem mass spectrometry (6). Bailey and Manoil reported a method for genome-wide tagging of bacterial proteins based on a broad–host range transpson which contain HA epitope and a hexahistidine sequence, which can be detected and the labeled proteins can be purified by metal affinity method (26). In the study of global protein localization of E. coli, a Japanese research group has done a lot of work, which is a branch of Genome Analysis Project. They inserted ORF of E. coli into N-terminal of GFPuv4 in a pCA24N plasmid and then transformed this plasmid into E. coli K-12 W3110 strain.The localization of inserted proteins are determined by observing fluorescence signal under fluorescence microscopy. The 4351 ORF of E. coli have been tagged and 3982 proteins are located. These proteins can be located in a whole cell, on a membrane, or at the nucleoid. There are some other proteins which form foci in the cell. All these localization data can be searched on the Genobase database (http://ecoli.naist.jp/gb6/WGB/intro.html) (27). Large-scale visualization of protein localization in E. coli has been dependent on transposon-mediated random internal tagging and plasmid-based overexpression of epitope-tagged proteins as described above. However, internal tagging can easily interrupt protein functions. Plasmid-based overexpression of proteins may saturate intracellular localization mechanisms, leading to abnormal localized proteins. Viewing all the protein tagging and localization work so far, no chromosomal tagging of prokaryotic proteome with GFP has been reported. For the best studied prokaryote, E. coli, 9 recombination between its chromosome and foreign linear dsDNA is a rare event, mainly due to the presence of linear-dsDNA exonuclease, thus becoming a bottleneck in tagging protein on chromosome of E. coli chromosome directly. But recently, with the development of a new technology, recombinogenic engineering, or recombineering methodologies (28) , things have changed. Recombineering mediates the efficient integration of transformed linear dsDNA, containing short regions homologous to the target locus, into the chromosome. Watt et al used recombineering method to position a selection of fluorescent protein genes immediately preceding the stop codon of a representative subset of CDSs of diverse function on the E. coli chromosome. These tagged proteins are expressed from their endogenous promoters. They were subsequently able to determine the intracellular localization of 21 (out of 23) E. coli proteins targeted. At the same time, the localization pattern and dynamic localization of some special proteins also can be traced and the relationship between localization and function is further proved (29). Although GFP is a useful reporter for E. coli protein localization, there are some limitations noted. As reported by Feilmeier et al, GFP can‟t be folded correctly in the periplasmic space, so for those proteins who located in the periplasmic space, GFP is not an ideal reporter (30). In addition to wet lab approach, some computational methods also are used in predicting protein subcelluar localization of E. coli, such as amino acid composition-based methods; integration of various protein characteristics methods; sequence homology-based methods and a mixed method combine one-versus-one (1-v-1) SVM model with a structural homology approach (6). 10 1.2. Essential genes and persistent penes Protein localization provides a useful way to study the function and interactions of proteins, As we all know, the amount of genes have been discovered is very large and they are not all essential to the life of an organism. From an economical and efficiency standpoint, protein localization should first focus on those genes important to the life of an organism. There are about 4270 genes in E .coli genome. The functions of these genes are different from each other. Some genes play important role in cell metabolism, like accd and asd, while other genes are involved in cell division, like ftsz and ftsk and still others that control the mobility of E. coli namely cheA, cheB, cheC genes. Among these 4270 genes of E. coli, part of them are important to the growth of E. coli and the cell will die once they are lost, thus,named the“essential genes” (31). On the other hand, nonessential genes refer to those that are not lethal to the cell when knocked out of them. It is found that those essential genes are more conserved than nonessential genes (32). There are several methods which have been used to determine essential genes, as in Gerdes et al who defined that 620 ORFs are essential in E. coli via the genetic footprint technique (31). The essential genes also can be designated by their function. For example, considering that the ribosomal proteins are indispensible to the growth of E. coli, those ribosomal genes are essential genes. But other genes, whose products play a role in cell motility or chemotaxis, are not as necessary to cell growth thus, thought to be nonessential genes. The essential genes are thought of as potential targets for antibiotics. The most commonly used methods on identifying essential genes for antibiotics targets are conditional mutagenesis. There are several approaches to conditional mutagenesis which include temperature-sensitive mutations (33), antisense mutants (34), change amber mutations to a temperature-sensitive phenotype by using temperature-sensitive tRNA (35), and/or using a promoter to control the target gene (36). To overcome the disadvantages of the above mentioned methods, Herring et al employed a new conditional mutation system 11 in E.coli, which is a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. They tried to make conditional lethal mutations in 8 essential genes of E.coli and 7 of them were successful (37). Different approaches may give different amount of essential genes. The essentiality of a gene is only meaningful in certain experimental conditions. Thus, the spectrum of essentiality changes in different growth conditions. For example, in laboratory conditions, bacteria usually grow on solid rich media.When cultured cells on rich media contain enough amino acids, nucleobases and vitamins, those genes in charge of synthesizing these product are not thought of as essential genes. But once transferred to a media lacking these compounds, the cell requires producing them de novo, and thus the same genes will be classified as essential genes. In addition to the media component, the physical growth conditions of the bacterial cell also change the essentiality spectrum, such as temperature, metal concentration, etc. So the „„essential‟‟genes should be carefully viewed as in the context (38). Different experiment approaches give different essential genes pool. Some experiment methods evenly are argued misestimate the amount of essential genes. It is quite different for a cell to survive in a laboratory compared to its natural setting when it competes for limited resources. Therefore, more genes are likely needed for a cell to survive naturally. Danchin et al focused on the persistence to explore gene essentiality by comparing 55 genomes of Firmicutes and Gamma-proteobacteria, which identified 611 genes in E. coil that are persistent among genomes, showing the characteristics of experimentally essential genes. In fact, the experimentally essential genes of E. coli constitute a subset of persistent genes with only a few exceptions. The results show that most of the persistent essential genes are involved in information transfer class, while most of the genes in charge of maintenance and stress response are persistent nonessential genes. This analysis strongly supports the hypothesis that wet lab experiment cannot detect all essential genes especially those only function in the situation of stress or starvation (39). 12 These results suggest that more research need to focus on these persistent genes. 1.3. Homologus recombination and recombineering Now the dominant wet-lab method in protein localization is labeling target genes with short peptides or report proteins. In plasmid-based protein localization method, the reporters are inserted into the N or C-terminus of target proteins in expression plasmid by restriction enzyme and ligase. But the most employed way in labeling target gene in chromosome directly is homologous recombination. 1.3.1 Homologous recombination Homology recombination is a biological process that is essential to all organisms. It uses sequence homology to promote DNA exchange, leading to new combinations of sequences. This biological process is conserved in all life forms, even in viruses. The functions of homology recombination are from sexual reproduction to generation of genetic diversity, and the maintenance of genomic integrity. Unveiling the detailed mechanism of homology recombination that all proteins use is important to understanding life. 1.3.1.1 Homologous recombination in E. coli Genetic recombination was discovered in E.coli by Lederberg and Tatum (40). In 1964, Robin Holliday describes the basic model of recombination (41). According to Holliday model, the first step is initiation. The recognition of one base sequence by another must be involved in this step in order to keep the accuracy of recombination. There are three possible interactions with the double-stranded DNA: single-strands with single-strands; single-strands with 13 double-strands, and double-strands with double-strands. These interactions results in the formation of heteroduplex DNA adjacent to one or more Holliday junctions. The amount of heteroduplex DNA will be increased or decreased by branch-migration when a Holliday junction is formed. The second step calls for the repair of mismatched information. Mismatch correction enzyme will work once mismatch has been generated within the heteroduplex DNA due to the fact that two recombining partners are not identical. The last step involves resolution. This step can be done in many ways based on the intermediate and the enzyme activities available. In this step, Holliday junction is resolved in two specific directions to produce recombinant progeny (42). There are many proteins involved in homology recombination, including RecA protein, RecBCD enzyme, RecFOR proteins, single-stranded DNA-binding protein (SSB protein), DNA polymerase I, DNA gyrase, and DNA ligase, as well as the RuvA, RuvB, and RuvC proteins or the RecG protein, etc (42). But not all these proteins are in need of the entire recombination process. The specific proteins involved are determined by both the genetic background of the strain and by the type of recombination event being monitored. In E. coli, homologous recombination runs by two pathways based on two different kinds of substrates (43). Although same proteins are involved in the migration and the resolution of a Holliday junction steps, there are different proteins found at the first two steps of homologous recombination at dsDNA ends and ssDNA gaps in E. coli. At dsDNA ends, RecBCD degrade DNA until it encounter a chi site, then a 3‟ended ssDNA to which it loads RecA is produced by helicase-nuclease activity of RecBCD. But at gaps, RecJ, the 5‟ ssDNA exounclease, extends the single-stranded DNA region and RecFOR (RecF, RecO, and RecR) promote RecA binding to SSB-coated DNA. The biochemical activities of the RecA protein have been extensively studied. The bacterial RecA is a highly conserved protein. RecA protein of 14 E.coli plays important roles in many biological processes, like induction of the SOS response to DNA damage, SOS mutagenesis, and recombinational DNA repair, +. In different biological processes, RecA play several roles. For example, in SOS induction, RecA binds to DNA form filament to facilitate an autocatalytic cleavage of the LexA repressor. In SOS mutagenesis, RecA is essential to stimulate DNA polymerase V. The kinetics of RecA binding to single-stranded DNA is more rapid than to double-stranded DNA. RecA protein promotes a DNA strand exchange reaction which is stimulated by the single-stranded DNA binding protein of E. coli. RecA is also a DNA-dependent ATPase, which is useful to recombination because some aspect of the DNA strand exchange reaction need ATP hydrolysis. Although ATP hydrolysis is not required to promoter the basic process of DNA strand exchange, it is necessary in some ways. ATP hydrolysis allows the reaction to go to completion and renders the DNA strand exchange reaction unidirectional (44, 45). In addition, ATP hydrolysis is required for any DNA strand exchange involving two duplex DNAs (45, 46). In addition to autoregulation by the C-terminus itself, RecA is also regulated by other proteins, like single-strand DNA binding protein (SSB) and RecFOR proteins. SSB plays a complex role in RecA reaction. On one hand, if SSB is allowed to bind to the DNA prior to RecA, RecA filament nucleation is inhibited (47, 48). But this inhibition can be overcome in the bacterial cell by two other proteins: RecO and RecR (49, 50).The RecFOR are essential to load RecA protein onto SSB-coated DNA at single-strand gap, and RecF play arole in targeting this process to the ends of ssDNA gaps (51, 52). In addition to RecA and its regulatory proteins mentioned above, there are some other proteins involved in recombination process, such as the RecBCD, which is an ATP-dependent exonucleaase and helicase. It binds to the ends of dsDNA and unwinds the strands as it moves to the centre. RecBCD also cleaves DNA when it passes though the DNA. RuvABC complex is involved in resolving Holliday junction in E. coli. The RuvA and RuvB proteins promote ATP-dependent branch migration of the holliday junction, while 15 RuvC introduces nicks in two DNA strands allowing resolution of the Holliday junction to recombinant DNA molecules (53). 1.3.1.2 Homologous recombination in eukaryotes One of the important properties that distinguish eukaryotes from prokaryotes is meiosis which plays important role in genetic exchanges and crossovers. Recombination not only occurs in mitotic cells from yeast to mammals but performs its highest frequency and complexity in meiosis (54). At least in the first meiotic division, recombination plays a crucial role in the segregation of chromosomes (55). There is at least one recombination event that happens for every pair of chromosome homologues in the nucleus. The event occurs more than five times for large human chromosomes. In meiosis, chromosome pairing is viewed and mediated by genome-wide searching for homology. Double-strand breaks (DSBs) happen in the early meiosis I phase in yeasts to promote chromosome pairing and recombination to proceed. Wu et al reported that the chromatin structure determine DSB site and a nonspecific enzyme cleaves it, which is different from χ site that is cleaved by a specific sequence (56). Another big difference between eukaryotes and prokaryotes in homology recombination is the enzymes involved in. As mentioned above, one of the essential enzymes of homology recombination in prokaryotes is RecA. However, in eukaryotes, two other proteins, Dmc1 and Rad51, play the same role as RecA. It has been reported that if only one mutation occurs with Dmc1 or Rad51, the recombination is reduced (57, 58). But, once the function of these two proteins are lost at the same time, defective in recombination will happen. Dmc1 is essential in the repair of meiosis-specific DSBs (58). Rad51 promotes homologous pairing and strand exchange between two DNA molecules to mediate homology recombination. Dmc1 and Rad51 share about 16 30% amino acid identities to RecA protein, with the difference at the amino and carboxyl termini region. In yeasts, Rad51 can bind to ssDNA and dsDNA (59) and it can mediate ATP-dependent homologous DNA pairing and strand exchange, like the roles of RecA in E. coli (60). Ristic et al found that Rad51 is dynamic with the active exchange of free and bound DNA- form (61). Other Rad51 like proteins also were identified in various eukaryotes which includes the product of the REC2 gene in Ustilago maydis (62), a RecA homologue from lilium and Rad51 homologue in mouse (63) and human cells (64). These findings support that the homology recombination and the main involved protein is a common phenomenon in nature. In eukaryotes, in addition to Dmc1 and Rad51, other proteins also can be found in Homology Recombination process. Rad52, a protein which binds to Rad51 (65), plays a role in promoting some reactions that controls the entry and exit of recombination substrates into different recombination pathways (66, 67). Some experiments confirmed that three other genes namely RAD54, RAD55, RAD57 are required to provide an approach to chromatin regions in order to make a combination available (68). In the process of Homology Recombination, strand transfer activities need some proteins to take part in it. Strand Exchange Protein 1 (SEP1) and Strand Transfer Protein (STPβ) are two proteins in eukaryotes which promote the formation of joint molecules from homologous linear duplexes and circular single strands. This process is Mg2+ requiring and ATP-independent (55). More experiment discloses that DNA damage signaling proteins, Nbs1 and ATR, also play roles in Homology Recombination of eukaryotes (69). Recently, a new tool based on the homologous recombination and three recombination proteins of λ phage has been developed and has been widely used in many study fields, like protein localization, gene function, etc. This technology is named Recombineering, Recombination-mediated Genetic Engineering. 17 1.3.2 Recombineering 1.3.2.1 Concept and mechanism of recombineering Recombineering, a new in vivo genetic engineering has been developed for use in E .coli. Due to its precision, efficiency, and simplicity, it may replace traditional in vitro cloning techniques. This technology works by generalized recombination function produced by phage proteins. It allows the efficient integration of transformed linear dsDNA (usually PCR generated), which contains short regions (ca. 35-55 bp) homologous to the target locus at both ends, into the chromosome without the necessity of restriction enzymes or DNA ligase. Recombineeing also can be used to clone or modify genes carried on plasmids, bacterial artificial chromosomes (BACs). Recombineering provides a method that permits rapid and precise in vivo manipulations of DNA and has become a new research tool in functional genomic analyses. There are two kinds of Recombineering based on the different phage proteins, which include RecE/RecT from Rac prophage and Red exo/beta/gamma from lambda bacteriophage (70, 71) with the latter being used usually. This introduction will focus mainly on Red exo/beta/gamma from lambda bacteriophage. There are three important proteins in Red exo/beta/gamma from lambda bacteriophage. The first is λ-Exo protein, which degrades linear dsDNA in a 5‟to 3‟direction from both ends. The degradation rate is around 10 to 30 bases per s in vitro (72-74), although has also been reported with a high rate of 1000 bases per s (75). The dsDNA ends are required for it to initiate digestion and remains bound to the dsDNA when it degrades one strand. If 5‟P is absent, the efficiency of the initiating reaction is low. In the presence of 5‟P, Exo binds to a dsDNA end and move along the 3‟ended single strand, resulting in a 3‟overhang (73, 76). In addition, λ-Beta is another enzyme essential to 18 Recombineering, which is a protein that binds to ssDNAs of >35 bp (77) and protect ssDNA from digestion by single-strand nucleases (78, 79). The λ-Beta protein also plays a role in promoting pairing or annealing between complementary ssDNAs (78-80). Some experiments confirmed that Exo and Beta form a complex in vivo (81, 82). It is not difficult to understand that when Exo degrades a dsDNA and generates an ssDNA overhang, then the Beta binds to this ssDNA overhang and pairs it with a complementary ssDNA target. A similarly model is also reported between RecBCD and RecA actions (83). It is essential for regulating the nuclease activity to limit DNA digestion to the needed amount for recombination that combine a specific exonuclease with its special annealing protein (84). Interestingly, this combination has homospecific, pairs of RecE-Beta or Exo-RecT will cause the recombination to fail (85). This may be caused by their protein-protein interaction specificities. RecBCD destroys any linear dsDNA and rolling-cycle replication products in the cell and inhibit recombination. In order to inhibit the activities of RecBCD, the third protein λ-Gam binds stoichiometrically to the RecBCD (86, 87). It is reported that λ-Gam also inhibits another enzyme, SbcCD endonuclease, which repairs double-strand breaks (88).This implies that λ-Gam inhibits enzymes involved in the double–strand-break-dependent recombination. But the problem in applying recombineeing to operate on plasmid caused by the characteristics of λ-Gam is that the inhibition of λ-Gam to RecBCD will produce abnormal rolling-circle replication of plasmids in the cell because RecBCD also destorys rolling-circle replication products (89). In conclusion, three enzymes, Exo, Beta and Gam, play different but important roles in recombineering. Exo and Beta are needed to handle the DNA prior to recombination, and Gam can protect the linear dsDNA from degradation by the RecBCD or SbcCD nuclease. 19 1.3.2.2 Application of recombineering Due to the high efficiency and short homology needed, recombineering has been widely used in molecular biology. It can be used in inserting foreign fragment into plasmid, bacterial chromosomal DNA or BACs; and it can be used to insert reporters (e.g.GFP,LacZ) or site-specific recombinases (e.g.Flp,Cre) or selectable markers (e.g. chloramphenicol gene) into chromosomal directly. It also can be used in making mutations in bacterial chromosomes directly (90). Watt et al labeled 23 genes of E. coli with Enhanced Green Fluorescent Protein (EGFP) and determined the intracellular localization of 21 proteins targeted by recombineering protocol (29). They also found that the PyrH protein localized dynamically to one cell pole and was asymmetrically segregated between daughter cells during cell division. Compared with other plasmid-based localization method, the advantage of recombineering label method is the expression of target - reporter fusion protein is under the control of the promoter of target genes, so reflection of the true picture of target protein would be guaranteed as far as possible. Due to its convenience and low cost, more researchers are applying recombineering to construct fusion protein library in bacterial. By recombineering technology, Butland and co-workers detected the expression of 648 chromosomally-encoded affinity-tagged CDSs, in their large-scale „tandem affinity purification‟ (TAP) protein interaction network analysis in E. coli, from a total of 857 successfully constructed strains. They found many new interactions and gave new clues to discover the function of previously uncharacterized proteins (91). Recently, Hu et al generated a network of 5,993 putative physical interactions among 1,757 E. coli proteins, including 451 function unannotated proteins, in E. coli by analysing the multiprotein complex isolated by an optimized Sequential Peptide Affinity (SPA)-tagging system based on recombineering technology. These protein interaction information are very useful to understand the function of these unannotated proteins (92). Recently, Taniguchi et al quantified the E. coli proteome and transcriptome by using libraries generated by recombineering technology (93). Recombineeing makes research work more efficient, for example, Chan et al constructed 96 20 conditional knockout targeting vectors simultaneously with recombineering technology (94). Although recombineering is gradually becoming a useful tool in bacterial research, it still has some limitations. First, the incorrect recombination between unwanted sequences can easily occur due to the short regions of homology that is needed for recombination. The second limitation is the foreign DNA fragment used in recombineering to be produced by PCR. Thus sequencing is important to ensure no mutation in the PCR cassette. Thirdly the recombineeing-based method is mainly used in few prokaryotes: E.coli, Salmonella (95), and Yersinia (96). Much research work is needed to apply this method to more and more prokaryotes. In the study of protein localization by tagging method, in addition to appropriate technology that is used to label target protein, another important consideration is what tag should be selected. As noted above, there are two kinds of tags that have been used in protein localization: short peptides and proteins. Both of these tags have their advantages and disadvantages. Now, fluorescent proteins, especially green fluorescent protein, have been widely used in the study of protein localization. 1.4 Green Fluorescent Protein (GFP) 1.4.1 Properties of GFP and its variants Bioluminescence is found in many organisms in nature from vertebrate to microorganisms and it plays important roles in the life of these organisms. Organisms use bioluminescence to help deter predators or help in predation or communicate with each other (97, 98). Organisms produce bioluminescence by luciferase (enzyme) and luciferin (substate).The latter can emit light when reaction with oxygen, and luciferase is a catalyst to speed up this reaction. With the discovery of the physical and biochemical characteristic of various 21 bioluminescences, it not only plays a role in the life of organisms, more being applied in biological research work. Bacterial lux genes, which encode the bacterial luciferase, have been used as reporters in many research works, such as visualizing gene expression in Streptomyces (99). Green Fluorescent Protein (GFP), one of the bioluminescence, becomes a star as a tool for biological research. It was first isolated from the jellyfish Aequorea Victoria (100). GFP converts the blue chemiluminescence of other proteins to green fluorescent light (101). The wild-type fluorescence absorbance/excitation peak is at 495nmwhile the normal emission peak of GFP is at 508nm (102). By computing and observing the absorption spectra of the fluorophore at different pH and other experiments, it is suggests that the protonated nitrogen composes an important factor in the function of GFP (103). There are other important characteristics which relate to the application in biological research identified by physical and chemical studies of purified GFP. The experiment shows that GFP is very resistant to denaturation (104); the elucidation of its 3D structure (105, 106); and the discovery of fluorescent proteins with varied emission and excitation or other origins based on the predicted changes in the structure that has expanded its application in biology. One of the advantages of GFP as an ideal reporter of gene expression is its high level of stability. The stability of GFP is determined by its special three-dimensional structure, which also presents as a new protein class, named β-can. In β-can, 11 β-strands surround and protect the chromophore that is positioned near the center of β-can. Both ends of the can are capped by small sections of loops and irregular α-helices (106). The structure of Aequorea GFP chromophore was first disclosed by Shimomura (107), which is a hexapeptide. It is reported that the hexapeptide between Renilla reniformis and Aequorea has similar identity except that Val68 and Gln69 are replaced by Asp and Arg, respectively. The chromophore of GFP consists of residues 65-67 (Ser - Tyr - Gly), the principle fluorophore of the proteinwhich does not remain static. Some chromophore variants of GFP have been isolated and has been reported that Serine at site 65 has been 22 replaced by Thr,Ala,Cys,Leu, and gly and that Tyrosine at site 66 has been replaced by Phe,Trp and His (108, 109). The excitation maximum of GFP is close to the ultraviolet range. Because ultraviolet light requires special optical considerations and can damage living cells, it is a disandvantage to apply GFP onto live cell imaging with fluorescence microscopy. To biology researchers, another important aspect of applying GFP to the cellular and other related research fields is the brightness of GFP. If the expression of GFP is controlled under a strong promoter, the fluorescence image is enough to observe. But, in most situations of biology research, the GFP is controlled by a weak promoter, often giving disappointing results. So developing brighter GFPs can help in the future success in the field of biology research. There are many ways in improving the brightness of GFP, like improving codon usage, removing GFP-containing inclusion bodies, etc. Presently, most research focus on overcoming the mentioned above two bottlenecks by using mutant amino acids of GFP. The mutation methods include random mutagenesis by chemical mutagens, error-prone PCR, deliberate site-directed mutations, DNA shuffling, directed evolution, etc (110). The excitation maximum of green fluorescent protein is successfully shifted to 488 nm by introducing a single point mutation altering the serine at position 65 into a threonine residue. Enhanced Green Fluorescent Protein (EGFP), a brighter Green Fluorescent Protein, has also been produced by mutation method. Cormack et al reported that they produce a yeast-Enhanced Green Fluorescent Protein (yEGFP) by optimizing encoding codons and introducing two amino acid changes (S65G; S72A) (111). By using Fluorescence-Activated Cell Sorting (FACS) select library of random amino acid mutations in the twenty amino acid flanking the chromophore Ser-Tyr-Gly sequence, Comack et al find variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type when excited at 488 nm (112). Based on these research results, a more extensive and effective application of GFP to biological research has been achieved. 23 Based on the knowledge of the GFP crystal structure and amino acids components of GFP fluorophore, several GFP variants that can produce different colors have been provided by corresponding amino acids mutation of GFP. The first point mutation of GFP (Ser65Thr) reported by Roger Tsien has higher fluorescence intensity and photostability than the widetype GFP (113). YFP, Yellow Fluorescent Protein, is a group of GFP variants with Thr203Tyr mutation. This substitution causes the most dramatic red shift (114). YFP has different versions, like mCitrine (115), Venus (116), and EYFP, etc. These YFP variants have different excitation wavelength, emission wavelength, brightness and photostability between each other (117). CFP, Cyan Fluorescent Protein, is a group of GFP variants with Tyr66Trp. CFP also has different versions, they include mCFPm (118), Cypet (119), etecetra. BFP, Blue Fluorescent Protein, is a group of GFP variants with Tyr66His (120). Different BFP has different brightness, for example, the double mutant Tyr66His/Tyr145Phe has almost the twice brightness of single mutant Tyr66His (121). The fouth GFP variant is Red Fluorescent Protein, like mCherry (122), DsRed, etc. These different GFP variants provide more choices in the study of proteins. But these Fluorescent Protein variants are most widely used in Fluorescence Resonance Energy Transfer (FRET) experiment, which used to study the interaction between proteins that are labeled with different fluorescent proteins (123). Based on the previous research result of our lab (29) and the resources we have, the EGFP is selected as tag in this project. 1.4.2 The application of GFP in prokaryotes The advantages of GFP allow it to be used as reporters of gene expression and dynamic processes during bacterial growth, and even the behavior of single bacteria in group and environments. In most research work using GFP as reporter in bacterial, multicopy plasmids containing GFP gene is employed (124, 125).Tombolini et al and other researchers also express GFP in Pseudomonas sp. by single copy fusions with strong promoters (126). In this work, they found that weaker fluorescence signal can be found during the 24 exponential growth phase in individual bacteria and maximal fluorescence signal is achieved until stationary growth phase. Another way it is used involves the insertion of GFP gene into the chromosome of bacteria directly. This method has its unique advantages in that it can paint a clearer picture of the target genes although sometimes the fluorescence signal cannot be detected due to the weak promoter of the target genes. With the development of new recombination technology, recombineering, plays a more important role in the study of gene function, protein localization and protein interaction of bacterial. In the field of bacterial development and cell biology, GFP has been widely used. Arigoni et al located SpoIIE of Bacillus subtilis, an important protein in triggering the development of progeny after cell division, to a area close to the pole of sporangia prior to septum formation by labeling it with GFP (127). Another example is SpoIVFB localization in Bacillus subtilis. SpoIVFB is proposed to be a proteolytic activator of a mother-cell-specific σk and previous immunofluorescece location method is difficult to apply to this protein because purifying adequate anti-SpoIVB antibodies is difficult. Resnekov et al overcome the problem and located the SpoIVB successfully by SpoIVB-GFP fusion method. The localization of SpoIVB support the model couples development in the forespore to mother cell transcription (128). In E. coli, GFP plays an important role in studying the localization and characteristics of two bacterial division proteins: FtsZ and FtsA. First, the localization of these two proteins is determined in living bacteria by using GFP fusions (129). FtsZ-GFP and FtsA-GFP localize to the division site. FtsA-GFP also can be found on membrane and at ring structure in the division plane at both early and late stages of septum formation. At the same time, Cormack et al visualize the formation of the division ring in actively dividing cells (112). The roles of N-terminal and C-terminal in the function of FtsZ also are disclosed by fusing different lengths of FtsZ to GFP (129). Thus, it is confirmed that FtsZ directly interacts with FtsA by introducing FtsA-GFP and FtsZ-GFP into the same cell. 25 Besides being applied to the study of proteins in cells, GFP is used in studying the interactions between bacterial and host. In this field, GFP has its advantages in stable fluorescence signal, reducing potential interference with surface contact between bacteria and host cells. Kain et al observe the rearrangement of host cell actin induced by S.tyhimurium invasion using GFP-tagged bacteria (130). GFP can be used to trace GFP expressing strain in the tissues of infected animals. Up to 5 weeks postinfection of M.marinum, Valdivia et al observed fluorescence signal of M.marinum expressing GFP in cryosections of chronically infected frog spleens (131). Another useful application of GFP in prokaryotes is the ability to separate bacteria on the basis of the relative fluorescence intensities. The name of this technology is Fluorescence-Activated Cell Sorting (FACS), a specialized type of flow cytometry. This technology allow determination of the levels of gene expression for every bacterial cell in the population and separate them on the basis of different levels of gene expression by reading the fluorescence intensity of each particle that passes through the laser sensing area. With FACS, a mixed population of mycobacteria containing transcriptionally weak and strong gene GFP fusions has been separated (125). In theory, any GFP containing bacteria with absolute fluorescence intensity can be specifically separated with FACS. Thus, FACS in combination with GFP can be used in different research experiments, such as identifying genes induced under different conditions. 26 1.4.3 The application of GFP in eukaryotes Green Fluorescent Protein (GFP) has also gained widespread application invarious research works involving yeasts, which includes the study of organelle structure, function and inheritance, the localization of proteins, DNA, RNA and liquid, and the disclosing of protein-protein interactions. GFP fusion proteins are valuable tools for intracellular compartments in studies of yeast organelle structure, function and inheritance. Organelles can be visualized with GFP in two ways: first with the fusing of GFP to the proteins that are located exclusively to the organelle, and second with the fusing of GFP to the signal peptides of the corresponding organelles. By labeling well-located ER protein Sec63P with GFP, Prinz et al studied the dynamic structure of ER in S.cerevisiae (132). To observe the structure and to determine factors influencing the mitochondrial morphology, a mitochondrial localized GFP (mtGFP) is constructed by fusing the corresponding localizing signal peptide at the N-terminal of GFP (133, 134). Localization research is another important area in which GFP comes into its own. In the field of yeast protein localization, it can be found that the application of GFP from static location to dynamic protein movement and trafficking to localization of individual protein to localization of global proteome. It is reported that the Msn2p-GFP and Msn4p-GFP are localized to the cytoplasm in normal situations but they will gather rapidly in the nucleus when the cell is under stress (135). As two stress-response transcription factors, it is not difficult to understand the localization pattern change of these two proteins. By combining the use of GFP and other GFP derivative YFP (Yellow Fluorescent Protein), CFP (Cyan Fluorescent Protein), various results have been achieved. Browning et al studied the in vivo movement of cargo protein Tea2p kinesin along microtubules in S.pombe by observing fluorescence signal of Tea2p-YFP and tubulin-CFP in the same cell with time-lapse fluorescence microscopy (136). By tagging DNA-bindings proteins or RNA-binding 27 proteins, the localization of corresponding DNA and RNA have been determined (137-140). A new technology, named Fluorescence Resonance Energy Transfer (FRET), became a useful tool for protein interactions research. During FRET, the excited donor fluorophore directly transfer energy to the acceptor fluorophore and the FRET can be detected by measuring emission from acceptor. The direct interaction between two tagged proteins can be determined with FRET because FRET only occurs when the donor and acceptor are very close in space. With FRET, Damelin et al identified the interaction between nuclear pore proteins and nuclear transport receptors (141). The characteristics of motif required for oligomerization of G-protein coupled receptor (GPCR) also can be demonstrated by using this method (142). 1.5 Aims of the project The aim of my study is to tag all the persistent genes of E. coli genome with EGFP by recombineering method. Specifically, I want to achieve the following: (1) Resources development: Generate EGFP-tagged E. coli persistent gene products library for systematic analysis of E. coli proteins. In this study, DY330 was chosen to construct the EGFP-tagged E. coli persistent gene products strain library since DY330 is essentially the wild type (W3110) except an integrated defective λ prophage. Successfully tagged strains will be prepared from independent colonies and stored in -80°C with glycerol stock. (2) Functional analysis: Characterization of the subcellular localization and dynamics of the E. coli proteome. Colonies for each of the successfully tagged CDS will be analyzed by fluorescence microscopy for subcellular protein localization. The successfully tagged CDS will be examined in at least two conditions: during the log or stationary phases. Some tagged strains will be selected to study dynamic protein expression and distribution in details. The 28 dynamic changes in expression and localization of these proteins will be monitored by video fluorescent microscopy throughout the cell cycle. (3) Bioinformatics: Establish an E. coli proteins localization database to provide a resources sharing platform for the scientific community. A database will be constructed and a web-based user interface will be designed for the database. The information of this database include tagging primer sequence, description, images, videos that are used to assign protein localizations in this study and the database will be integrated with existing E. coli database. In summary, this project is the first for genome-scale determination of prokaryotic protein subcellular localization using chromosomal EGFP-tagging. The determination of the dynamic subcellular localization of the E. coli proteome is vital for the understanding of prokaryotes. By accomplishing the objectives of this project, the experimental and informational resources founded in the study will be a helpful tool in the scientific community for the complete understanding of the structure and function of the living cell. 29 Chapter 2: Materials and Methods 2.1 Materials Sequence data, gene annotations and associated information were obtained from the Colibri (http://genolist.pasteur.fr/Colibri/) and Genobase (http://www.ecolihub.org/GenoBase) databases. The list of tagged genes can be found in Appedix 2. 2.1.1 Bacterial strains E. coli DY330 (W3110_lacU169gal490 [λcl857_ (cro–bioA)]) was used for these experiments. 2.1.2 Plasmid and primers The source of the fluorescent domain was the EGFP (Enhanced Green Fluorescent Protein) coding sequence. The plasmid used for producing the tagging cassettes was pEGFP-loxp-Cm-loxp (Figure 1). All oligonucleotides were purchased from Techdragon (Hong Kong), Invitrogen (Hong Kong) and Sunbiotech (Beijing) as the salt-free form. Linear dsDNA targeting cassettes were synthesized by PCR using the appropriate pair of primers (forward primers also encoded a short peptide linker (-LEGSG-) to separate the CDS and fluorescent protein domains) and plasmid templates (pEGFP-loxP-Cm-loxP). All primers used in this project are list in Appendix 3. 30 Figure 1 Map of plasmid used as PCR template for producing the tagging cassette. This plasmid is constructed by inserting loxp-Cm-loxp sequence downstream of EGFP gene of commercial pEGFP plasmid (BD bioscience). Forward primer used to produce linear dsDNA targeting cassettes is complimentary to N-terminal of EGFP and reverse primer used to produce linear dsDNA targeting cassettes is complimentary to the second loxp sequence. This plasmid map is created using PlasMapper. 31 2.1.3 Agarose, Antibiotic and Culture Medium LB (Luria-Bertani) medium: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl . pH was adjusted to 7.0 with 5 M NaOH . LB (Luria-Bertani) agarose medium: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 15 g/L agar. pH was adjusted to 7.0 with 5 M NaOH. The stock concentration of antibiotic are: Chloramphenicol, 50 mg/ml in methonal; Ampicillin, 50 mg/ml; Kanamycin, 10 mg/ml. Antibiotic stock solutions were aliquot into microfuge tubes and kept at -20°C. Antibiotic agar plate: CM plate, 12.5 μg/ml Chloramphenicol; Amp plate, 50 – 60 μg/ml Ampicillin; Kan plate, 15 μg/ml Kanamycin. 2.2 Experiment Methods 2.2.1 Preparation of component cell for plasmid amplification and plasmid transformation 400 μl overnight cultures (2-3ml LB medium inoculated from single colonies, grown at 37°C for 18 h at a speed of 220 rpm) was expanded into 45 ml of LB medium in a 250 ml Erlenmeyer flask, and incubated at 37°C for 2–3h (until OD600 of ca. 0.4–0.6) at a speed of 220 rpm. Flasks were cooled to 0°C as rapidly as possible in iced water. After 15–20 min, cells were harvested by centrifugation at 0°C (4000 g, 5 min). Cell pellets were carefully washed three times with sterilized ice-cold water (2×50 ml, then 1×1.5 ml) then re-suspended in 200 ml of ice-cold water. Competent cells (50 ml) were transformed with 50–200 ng of plasmid using a BioRad electroporator (1.8 kV, 25 mF, 200 W). LB medium (1 ml) was added to the transformed cell mixture, which was incubated at 37°C, for 1-2 h. Cells were collected by centrifugation, supernatant media was discarded, and then the resuspended cells were plated onto LB agar containing the appropriate antibiotic to select for resistant colonies. 32 2.2.2 Plasmid DNA purification Plasmid extraction is carried out by using QIAprep® Miniprep kit of Qiagen. A single colony was picked from a freshly streaked selective plate and inoculated a culture of 5 ml LB medium containing the appropriate selective antibiotic. Incubate for 16 h at 37°C with vigorous shaking. The bacterial cells were harvested by centrifugation at 4000 rpm in a conventional microcentrifuge for 5 min at room temperature. Pelleted bacterial cells were reuspended in 250 μl Buffer P1, and then the mixture was transfered to a microcentrifuge and mixed thoroughly by inverting the tube 4–6 times. 350 μl Buffer N3 was added, and then mixed immediately and thoroughly by inverting the tube 4–6 times.This mixture was centrifuged for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. The supernatants were applied to the QIAprep spin column by decanting or pipetting and centrifuged for 30–60 s. The flow-through was discarded. The QIAprep spin column was washed by adding 0.5 ml Buffer PB and centrifuged for 30–60 s. The flow-through was discarded. QIAprep spin column was washed by adding 0.75 ml Buffer PE and centrifuged for 30–60 s. The flow-through was discarded, and centrifuged for an additional 1 min to remove residual wash buffer. The QIAprep column was placed in a clean 1.5 ml microcentrifuge tube. To elute DNA, 30μl water was added to the center of each QIAprep spin column, let stand for 1 min, and was centrifuged for 1 min. 2.2.3 Amplification of linear dsDNA targeting cassettes by Polymerase Chain Reaction (PCR) Each PCR reaction included 0.5μl Expand Hi-Fidelity DNA polymerase (Roche) and 1×PCR buffer, 300mM of each dNTP, 5 ng of plasmid template, 300nM of each primer and sterilized water to 50μl. The typical PCR program used was 95°C for 60s, 5 cycles of (95°C for 20 s, 56°C for 20 s, 72°C for 180 s), 25 cycles of (95°C for 15 s, 52°C for 15 s, 72°C for 120s), 72°C for 720 s. PCR products were excised from 1.0% agarose gels (in 1×TAE) and purified 33 using a Qiagen Gel Extraction kit, eluting with 50 ml of double distilled H2O. For some PCR programs, an additional step of DpnI digestion was performed prior to gel purification. A 50 μl PCR reaction typically yielded enough DNA for one chromosomal targeting experiment. 2.2.4 PCR product purification PCR product purification was carried out by using QIAquick® spin kit of Qiagen. The DNA fragment was excised from the agarose gel with a clean, sharp scalpel. The gel slice was weighed in a colorless tube. 3 volumes of Buffer QG were added to 1 volume of gel (100 mg ~ 100 μl). The mixture was incubated at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, the mixture was mixed by vortexing the tube every 2–3 min during the incubation. After the gel slice has dissolved completely, 1 gel volume of isopropanol was added to the sample and mix (when DNA fragments are <500 bp and >4 kb). A QIAquick spin column was placed in a provided 2 ml collection tube. To bind DNA, the sample was applied to the QIAquick column, and centrifuged for 1 min. The flow-through was discarded and QIAquick column was placed back in the same collection tube. 0.5 ml of Buffer QG was added to QIAquick column and the QIAquick column was centrifuged for 1 min. To wash, 0.75 ml of Buffer PE was added to QIAquick column and the QIAquick column was centrifuged for 1 min. The flow-through was discarded and the QIAquick column was centrifuged for an additional 1 min at 17,900 x g (13,000 rpm). The QIAquick column was placed into a clean 1.5 ml microcentrifuge tube. To elute DNA, 30 μl water (pH 7.0–8.5) was added to the center of the QIAquick membrane and the QIAquick column was centrifuged for 1 min. 2.2.5 Restriction enzymes digestion of plasmids of purified PCR product For some reasons, plasmid can‟t removed fully by PCR product purification step. So DpnI digestion is necessary to every purified PCR product. The 34 digestion reaction system including 17 μl purified PCR product, 1μl DpnI enzyme (New England Biolabs Inc), 2μl 10×NEBuffer 4. Incubate at 37°C for 3 hours. Then purify the enzyme digested PCR product using QIAquick® spin kit of Qiagen. The purified PCR products are ready for transforming to DY330 component cell. 2.2.6 λ-Red mediated dsDNA recombination protocol 400 μl from overnight cultures [2-3ml LB medium inoculated from single colonies, grown at 32°C for 18 h at a speed of 220 rpm] was expanded into 45 ml of LB medium in a 250 ml Erlenmeyer flask, and incubated at 32°C for 2–3 hours (until OD600 of ca. 0.4–0.6) at a speed of 220 rpm. Flasks were transferred to a shaking water bath at 42°C and incubated for 14–15 min, before cooling to 0°C as rapidly as possible in iced water. After 15–20 min, cells were harvested by centrifugation at 0°C (4000 g, 5 min). Cell pellets were carefully washed three times with sterilized ice-cold water (2×50 ml, then 1×1.5 ml) then re-suspended in 200 ml of ice-cold water. Competent cells (50 μl) were transformed with 50–200 ng of (gel purified) linear dsDNA targeting cassette using a BioRad electroporator (1.8 kV, 25 mF, 200 W). LB medium (1 ml) was added to the transformed cell mixture, which was incubated at 32°C, for 1-2 hours. Cells were collected by centrifugation, supernatant media was discarded, and then the resuspended cells were plated onto LB agar containing the appropriate antibiotic [chloramphenicol (Cm, cat)] to select for resistant colonies. 2.2.7 PCR screening of recombinant clones Colony PCR was used to confirm the correct integration of the fluorescent protein gene and the adjacent „floxed‟ antibiotic-resistance gene, using two pairs of primers to separately amplify the 3'- and 5'- junctions with the chromosome. Each PCR reaction included 1μl ExTaq polymerase and 1× PCR buffer, 300 mM of each dNTP, 300nM of each primer and sterilized water to 50μl. The reaction program is 95°C for 180 s, 30 cycles of (95°C for 60 s, 35 55°C for 60s, 72°C for 120s) and 72°C for 600 s. PCR products were checked by running 1.0% agarose gels (in 1×TAE) and corresponding clones with positive PCR results were selected as positive tagged clones. 2.2.8 Fluorescence microscopy Fluorescent microscopy was performed with a Leica microscope using an oil immersion ×100 objective lens, a Spot CCD camera and Spot Advance software. EGFP fluorescence was measured as excitation / emission at 450-490 / 520 nm. The exposure time is different from every labeled protein. In total, 4 seconds exposure was sufficient to capture a strong fluorescent signal, but observing labeled proteins of weak fluorescence from more than 8 second exposure may be necessary. The fluorescence images were recorded and analysed using Advancespot and Photoshop software. 2.2.8.1 Cell Culture Condition Wild Type DY330 cells and EGFP-tagged DY330 cells were cultured in LB Broth (USB), EGFP-tagged DY330 cells were also cultured in LB Broth (USB) supplemented with 12.5μg/ml Chloramphenicol. Wild Type and EGFP-tagged DY330 cells were collected at different growth phases. 32°C was used for all cultures unless otherwise indicated to avoid the activation of the three recombineering related genes exo, beta and gam. 2.2.8.2 Staining chromosome with DAPI Cells treated with DAPI (1:1 with 1mg/ml DAPI) before being immobilized on glass slides coated with a partially dehydrated aqueous 1% agarose pad immediately prior to analysis. The culture was examined as excitation / emission wavelength 365 / 397 nm. The chromosome was stained bright blue with DAPI. 2.2.9 Time–lapse experiment For time-lapse microscopy observations, a glass slide filled with LB Broth 36 (USB) containing 1% agarose was used. Samples (5µl) of culture were removed during the mid-log phases and immobilized on a slide. Cells were visualized and photographed using a microscope (Zeiss) equipped with a camera. The photos are taken at 5-minute intervals for 120 minutes. System control and image processing were performed using supplied software (Ultraview ERS software, Perkin Elmer). 37 Chapter 3: Results 3.1 Construction of an EGFP-tagged library of persistent genes 3.1.1 Preparation of the tagging cassette by PCR Six hundreds and eleven persistent genes within E. coli were systematically tagged using λ-Red mediated recombineering technology (15 of them have been tagged and located previously in our lab by Watt et al (29)). For each gene, a pair of oligonucleotides was synthesized that included 51 bases homologous to the regions immediately up or downstream of the desired chromosomal insertion site, adjacent to 21 bases homologous to the plasmid template (which included an EGFP gene and antibiotic resistance cassette as a selectable marker). A five amino acid spacer coding sequence was positioned between the target gene and fluorescent protein coding domains, to ensure that they were spatially-separated, thus reducing the possibility of steric interference with the native function of the target protein. The tagging cassettes were amplified by PCR with template, plasmid pEGFP, and primers mentioned above. Correct PCR products were gel purified with Gel Cleanup Kit. The original and purified PCR products were tested by running 1.0% agarose gels. The length of the PCR product (tagging cassette) is about 2.3kb, including two homologous arms at each end of the cassette, an EGFP gene, a chloramphenicol-resistance gene which is flanked by two loxp sites. Due to the length of homologous arms being the same to each target gene, there is no difference between various target genes on the length of PCR product. Representative examples for frR, ftsI and spoT genes are shown in Figure 2. Restriction enzyme DpnI was used to digest the potential remains of plasmid template in purified PCR products. Not all tagging cassette of target genes are produced by PCR successfully. Some tagging cassette producing PCR failed, probably due to the incorrect PCR primers or PCR reaction conditions (Appendix 2). 38 M 1 2 3 Figure 2 Purified PCR product of targeting cassette. PCR primers each included 51 bases homologous to the chromosome immediately upstream and downstream of the desired insertion point. To every target gene, the main sequences of targeting cassette is the same, only the sequence of 51 bases homologous arm is different between different target genes. M: 1Kb DNA ladder 1-3: Purified PCR product of targeting cassette for labeling three different genes: frR, ftsI, spoT. 39 3.1.2 Tagging CDS of persistent genes in chromosomal and positive clone selection PCR products (dsDNA targeting cassettes) for each persistent gene were electroporated into E. coli DY330 according to published protocol (143). In general, 10-20 colonies can be found on selective plates after incubating for 24 hours at 32℃. 4-6 colonies were selected for each strain, and were screened by colony PCR using primers that flank the junctions created with the chromosome, to confirm that the exogenous cassette (EGFP loxP-Cm-loxP) had been accurately inserted at the 3' end of the target genes (Figure 3, Figure 4). There are two pair of primers and three PCR reactions employed in screening. The first pair of primers is forward primer 1 and reverse primer 2 with the forward primer 1 complimentary at the 3‟ end of the target gene and reverse primer 2 complimentary at the 5‟ end of the EGFP gene. This pair of primer is used to amplify the junction region of the target gene and EGFP gene. The second pair of primers is forward 3 and reverse primer 4 with the forward primer 3 complimentary at the 3‟ end of the EGFP gene and reverse primer 4 complimentary at the 5‟ end of the intracistronic region of the target gene. This pair of primers is used to amplify the junction region of the EGFP loxP-Cm-loxP cassette and the intracistronic region of the target gene. In order to confirm the whole tagging cassette is inserted into the chromosome correctly, the full tagging cassette is also amplified with forward primer 1 and reverse primer 4. The length of theses primers is from 19mers to 26mers. The positive clones are confirmed only when all these three PCR products are the same as expected. In general, the efficiency of tagging is high and one positive clone can be picked out of every third clones on selective plates. Stocks of each of the successfully tagged strains were prepared and stored at -80C after adding glycerol to 20%. In total, 189 genes can be tagged successfully, meaning the positive colonies can be selected. 422 genes are tagged failed, this including 194 genes that no colonies can be found on selective plates after recombineering. All tagging results are list as Appendix 2. 40 Figure 3 The strategy used to create chromosomal C-terminal fluorescent fusion proteins using a combination of λ-Red mediated homologous recombination and Cre/LoxP mediated site-specific recombination, as exemplified by the construction of suhB-EGFP. (Modifed diagram of Watt et al (29)) 41 M 1 2 3 Figure 4 PCR screening of correct recombinant spoT EGFP tagged clones. Colony PCR was used to confirm the correct integration of the target cassette which contains fluorescent protein gene and adjacent antibiotic-resistance gene, using two pairs of primers to separately amplify the 3‟and 5‟junctions with in the chromosome. M: 1Kb DNA ladder 1: Confirm PCR product with spoT Sense Primer (forward primer 1) complimentary at the 3‟ end of the spoT gene and EGFP 5'-Reverse Primer (reverse primer 2) complimentary at the 5‟ end of the EGFP gene. 2: Confirm PCR product with EGFP 3'-Forward Primer (Forward primer 3) complimentary at the 3‟ end of the EGFP gene and spoT Antisense Primer (reverse primer 4) complimentary at the 5‟ end of the intracistronic region of the spoT gene. 3: Confirm PCR product with spoT Sense Primer (forward primer 1) complimentary at the 3‟ end of the target gene and spoT Antisense Primer (reverse primer 4) complimentary at the 5‟ end of the intracistronic region of the spoT gene. 42 3.2 Subcellular localization of E. coli persistent proteins and subcellular localization of ribosomal subunit proteins of E. coli One hundred and eighty-nine genes were successfully tagged with the EGFP coding sequence, representing 31% of the 611 persistent CDSs in E. coli (Table 2). Strains containing tagged genes were inoculated into Luria Broth (LB) and log phase or stationary phase cells were collected and immobilized on agarose pads on glass sides for fluorescence microscopy. 139 of the EGFP fusion proteins could be located in cultures of the 189 successfully-constructed E. coli strains grown under these conditions. The localization pattern fall into three categories: cytoplasm, membrane, and focus (foci) at pole(s). Typical examples of every localization pattern are shown in Figure 5 and all fluorescence images of successfully located proteins are given as Appendix 1. Combined with the previous protein located results in our lab by Watt et al (29), the majority of these (115) were located in the cytoplasm, while 12 (~9% of the localized proteins) were concentrated predominantly or exclusively at one or two foci within the cytoplasm, and 8 (~6%) were positioned predominantly at the membrane (Table 3, Table 4). No fluorescence signal could be detected for the products of the remaining 50 tagged genes. Compared with previous reported E. coli protein localization results, most proteins show the same localization patterns in this study. But different localization results are also found. Some ribosomal subunit proteins have unexpected localization. 53 ribosomal subunit proteins have been tried to tag. RpsO, which encodes the 30S ribosomal subunit protein S15, was found to localize predominantly within foci at both poles of the cell. This is quite different from the diffuse intracellular distribution previously reported for RpsO (27). Six other ribosomal subunit proteins (RpsP, RpsQ, RpsU, RplC, RplT, and RplW) had polar localization patterns analogous to RpsO, but the other 1 ribosomal subunit proteins examined had diffuse (cytoplasmic) distributions (Table 5). In order to further investigate the subcellular 43 localization pattern of ribosomal subunit proteins, RpsO is selected as research object to do further research. It was reported that some proteins localize to different subcellular location in different growth phases (144). I also found that the localization pattern of SuhB protein is different at various growth phases (Fig 7). These observations prompt me to study how the RpsO protein moves in different phase of an E. coli cell cycle. First, RpsO-EGFP strains are cultured and collected at different growth phase (i.e. lag phase, log phase, and stationary phase) and are observed under a fluorescence microscope. It is found that there is no change of the localization pattern of RpsO at these three cell growth phases. Then the intracellular distribution of RpsO-EGFP was monitored during the process of cell division (i.e. throughout the cell cycle) by capturing time-lapse images every few minutes, to see whether it exhibited variable localization patterns. Initially RpsO-EGFP accumulated within focal points at each cell pole, and then another focal point formed at the mid-cell during the initiation of cytokinesis. After cell division was completed, two RpsO-protein focal points formed at the poles of each of the daughter cells. As the next cell division step began, an RpsO focal point again formed at the mid-cell (Figure 6). 44 Table 2 Experimental results of genes tagging and protein localization Category Proteins Tagged and located successfully Total 139 189 Tagged successfully but no fluorescence signal 50 Unsuccessful tagging due to colonies not being found on the selective plates 194 422 Unsuccessful tagging due to failure of confirmatory † PCR or other reasons 228 ‡ 611 †: Other reasons: False positive results due to plasmid contamination, tagging cassette production PCR failure, etc. ‡: These genes includes 15 genes that have been tagged and located previously in our lab by Watt et al (29) Table 3 E. coli fluorescent proteins localization Localization Proteins Cytoplasm 115 Membrane 8 Total 139 †: Foci at pole(s) or center 12 Cytoplasm or Foci 4 † Includes 13 genes that have been tagged and located previously in our lab by Watt et al (29) 45 Table 4 List of localization pattern of the succeed localized proteins CDS Tag Strain Description/Function Cellular Localization D-methionine transport abC EGFP DY330 Diffuse(weak) ATP-bindingprotein Acetyl-coenzyme accD aceF EGFP EGFP DY330 DY330 A carboxylase carboxyltransferase subunit beta Dihydrolipoyllysine-residue acetyltransferase Two foci at two poles Diffuse Component of pyruvate dehydrogenase complex # DsRed2 EL250 mRFP1 DY380 Adenylate kinase adK apaH EGFP DY330 Bis(5'-nucleosyl)-tetraphosphatase, symmetrical aroB EGFP DY330 3-dehydroquinate synthase aroC EGFP DY330 Chorismate synthase Diffuse One focus at pole (Log phase); Diffuse(Stationary phase) Diffuse Diffuse F1 sector of membrane-bound atpG EGFP DY330 ATP synthase Membrane gamma subunit bioA EGFP DY330 Adenosylmethionine-8-amino-7-oxonon anoate aminotransferase bioB EGFP DY330 Biotin synthase Diffuse Two foci at two poles bioF EGFP DY330 8-amino-7-oxononanoate synthase 46 Diffuse carA EGFP DY330 Carbamoyl phosphate synthetase small subunit Diffuse carB EGFP DY330 Carbamoyl-phosphate synthase large subunit Diffuse ccmA EGFP DY330 Cytochrome c biogenesis ATP-binding export protein ccmB EGFP DY330 Heme exporter protein B Membrane ccmC EGFP DY330 Heme exporter protein C Membrane ccmF EGFP DY330 Cytochrome c-type biogenesis protein Membrane clpB EGFP DY330 Protein disaggregation chaperone crcB EGFP DY330 Conserved hypothetical protein; putative inner membrane protein associated with chromosome condensation cysS EGFP DY330 Cysteinyl-tRNA synthetase deF EGFP DY330 Peptide deformylase Diffuse DNA polymerase III alpha subunit Diffuse EGFP DY330 EGFP DY330 Disulfide bond formation protein B Diffuse EGFP DY380 1-deoxy-xylulose phosphate synthase Diffuse efP EGFP DY330 Elongation factor EF-P Diffuse enO EGFP DY330 Enolase Diffuse dnaE dsbB # dxS # erA folB Diffuse(weak) Diffuse(weak) Membrane Diffuse(weak) EGFP DY380 Ras-like GTP-binding protein Diffuse EGFP DY330 Dihydroneopterin aldolase Diffuse folD EGFP DY330 Bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase and 5,10-methylene-tetrahydrofolate cyclohydrolase folK EGFP DY330 2-amino-4-hydroxy-6-hydroxymethyldi hydropteridine pyrophosphokinase frR EGFP DY330 Ribosome recycling factor EGFP DY330 Ferric uptake regulation protein Diffuse Diffuse Diffuse(weak) Diffuse fuR 47 gidB EGFP DY330 glnS EGFP DY330 Glutaminyl-tRNA synthetase Diffuse DY330 Serine hydroxymethyltransferase Diffuse DY330 Cpn10 chaperonin GroES, small subunit of GroESL glyA groS EGFP EGFP Methyltransferase Diffuse One focus at pole hemE EGFP DY330 Uroporphyrinogen decarboxylase Diffuse hemH EGFP DY330 Ferrochelatase Diffuse hflK EGFP DY330 HflA complex cleaves lambda cII Diffuse Diffuse hisA EGFP DY330 N-(5'-phospho-L-ribosyl-formimino)-5amino-1-(5'phosphoribosyl)-4-imidazolecarboxamid e isomerase hslU EGFP DY330 ATP-dependent hsl protease ATP-binding subunit Diffuse(weak) hslV EGFP DY330 ATP-dependent protease Diffuse(weak) htpG EGFP DY330 Chaperone Diffuse hupB EGFP DY330 DNA-binding protein HU-beta Diffuse ileS EGFP DY330 Isoleucyl-tRNA synthetase infB EGFP DY330 Translation initiation factor IF-2 Diffuse kdsB EGFP DY330 3-deoxy-manno-octulosonate cytidylyltransferase Diffuse ksgA EGFP DY330 Dimethyladenosine transferase Diffuse leuS EGFP DY330 Leucyl-tRNA synthetase Diffuse loN EGFP DY330 ATP-dependent protease Diffuse lpxA EGFP DY330 Acyl-[acyl-carrier-protein]--UDP-N-ace tylglucosamine O-acyltransferase Diffuse lpxC EGFP DY330 UDP-3-O-acyl N-acetylglucosamine deacetylase Diffuse 48 Diffuse(weak) maP # metF EGFP DY330 Methionine amino peptidase Diffuse EGFP DY330 5,10-methylenetetrahydrofolate reductase Diffuse EGFP EGFP metK # EYFP ECFP DsRed2 mRFP1 miaA EGFP EL250 DY330 DY380 DY380 Diffuse EL250 EL250 DY330 mpl EGFP DY330 mreC EGFP DY330 murB EGFP DY330 EGFP DY330 EGFP DY380 ECFP DY380 mRFP1 DY380 EGFP DY330 # 1-5 foci S-adenosyl-methionine synthetase nusA Delta(2)-isopentenylpyrophosphate tRNA-adenosine transferase UDP-N-acetylmuramate:L-alanyl-gamm a-D-glutamyl-me so-diaminopimelate ligase cell wall structural complex MreBCD transmembrane component MreC UDP-N-acetylenolpyruvoylglucosamine reductase Transcription elongation protein NusA Diffuse Diffuse Diffuse Diffuse 2-4 clusters (co-incident with nucleoids) Diffuse Diffuse pepA Aminopeptidase A (weak) Diffuse pepN EGFP DY330 Aminopeptidase N (weak) pgK EGFP DY330 Phosphoglycerate kinase 49 Diffuse ppA EGFP DY330 Inorganic pyrophosphatase prfC EGFP DY330 Peptide chain release factor RF-3 Diffuse prlC EGFP DY330 Oligopeptidase A Diffuse prmA EGFP DY330 Ribosomal protein L11 methyltransferase Diffuse proA EGFP DY330 Gamma-glutamylphosphate reductase Diffuse proC EGFP DY330 Pyrroline-5-carboxylate reductase Diffuse psD EGFP DY330 Phosphatidylserine decarboxylase proenzyme Diffuse ptH EGFP DY330 Peptidyl-tRNA hydrolase Diffuse pykA EGFP DY330 Pyruvate kinase II EGFP DY380 EGFP DY330 EYFP DY380 ECFP DY380 DsRed2 mRFP1 DY380 pyrH # Uridine monophosphate kinase One focus at pole Diffuse(weak) 1-4 foci (some highly mobile) Diffuse DY380 queA mRFPm ars DY380 RFP DY380 EGFP DY330 S-adenosylmethionine:tRNA ribosyltransferase-isomerase Diffuse (weak) One focus at pole recA EGFP DY330 General recombination Four-five foci at and DNA repair protein poles or at center # recG EGFP DY330 ATP-dependent DNA helicase recJ EGFP DY330 Single-stranded-DNA-specific exonuclease Diffuse rhO EGFP DY330 Transcription termination factor Diffuse ribC EGFP DY330 Riboflavin synthase alpha subunit Diffuse ribD EGFP DY330 Riboflavin biosynthesis protein Diffuse 50 Diffuse rluD EGFP DY330 Ribosomal large subunit pseudouridine synthase D rnE EGFP DY330 Ribonuclease E Membrane rnhA EGFP DY330 Ribonuclease HI Diffuse(weak) rnR EGFP DY330 Ribonuclease R Diffuse rpE EGFP DY330 Ribulose-phosphate 3-epimerase Diffuse rpH EGFP DY330 Ribonuclease PH Diffuse rpiA EGFP DY330 Ribose-5-phosphate isomerase A Diffuse rplC EGFP DY330 Diffuse Two foci at 50S ribosomal subunit protein L3 two poles Two foci at rplT EGFP DY330 50S ribosomal subunit protein L20 two poles rplU EGFP DY330 50S ribosomal subunit protein L21 Diffuse rplW EGFP DY330 50S ribosomal subunit protein L23 Two foci at two poles rpoH EGFP DY330 RNA polymerase sigma-32 factor Diffuse rpoZ EGFP DY330 DNA-directed RNA polymerase omega chain Diffuse rpsO EGFP DY330 30S ribosomal subunit protein S15 Two Foci at two poles Two Foci at rpsP EGFP DY330 30S ribosomal subunit protein S16 two poles Two Foci at rpsQ EGFP DY330 30S ribosomal subunit protein S17 two poles Two Foci at rpsU EGFP DY330 30S ribosomal subunit protein S21 two poles 51 rsmB EGFP DY330 Ribosomal RNA small subunit methyltransferase B secB EGFP DY330 Protein-export protein Diffuse serA EGFP DY330 D-3-phosphoglycerate dehydrogenase Diffuse serC EGFP DY330 Phosphoserine aminotransferase Diffuse(weak) serS EGFP DY330 Seryl-tRNA synthetase Diffuse(weak) smF EGFP DY330 Smf protein Diffuse(weak) smpB EGFP DY330 SsrA-binding protein spoT EGFP DY330 Guanosine-3',5'-bis(diphosphate) 3'-pyrophosphohydrolase sspA EGFP DY330 Stringent starvation protein A Diffuse suhB EGFP DY330 Inositol-1-monophosphatase Foci (Lag phase) Diffuse (Stationary phase) talB EGFP DY330 Transaldolase B Diffuse(weak) tktA EGFP DY330 Transketolase 1 Diffuse tpiA EGFP DY330 Triosephosphate isomerase Diffuse EGFP DY380 Probable tRNA modification GTPase Diffuse trpA EGFP DY330 Tryptophan synthase alpha chain Diffuse trxA EGFP DY330 Thioredoxin 1 tufB EGFP DY330 Protein chain elongation factor EF-Tu Diffuse usG EGFP DY330 Stabilizes phage lambda protein N-NusA-RNAP antitermination comple Diffuse DY380 Integral inner membrane metallo-protease trmE Diffuse(weak) Diffuse Diffuse(weak) # Diffuse(weak) Diffuse # yaeL EYFP Beside membrane yaeN EGFP DY330 Putative cell cycle protein mesJ Diffuse ybeB EGFP DY330 Hypothetical protein Diffuse 52 ydaO EGFP DY330 Hypothetical protein Diffuse ydhH EGFP DY330 Hypothetical protein Diffuse yfgB EGFP DY330 Hypothetical protein Membrane EYFP DY380 GTP-binding protein EngA Diffuse EGFP DY330 Hypothetical tRNA/rRNA methyltransferase Diffuse EYFP DY380 yfgK # yfhQ yfjB # Diffuse NAD kinase Some clusters ygcA EGFP DY330 23S rRNA (Uracil-5-)-methyltransferase ygfB EGFP DY330 Hypothetical protein Diffuse yggW EGFP DY330 Hypothetical protein Diffuse ECFP DY330 # ygjD Diffuse (weak) Diffuse Probable O-sialoglycoprotein peptidase EGFP DY380 yhbC EGFP DY330 Hypothetical protein yhbY EGFP DY330 Putative RNA-binding protein yhbZ EGFP DY330 Putative GTP-binding factor yhdG EGFP DY330 tRNA-dihydrouridine synthase B yihA EGFP DY330 GTP-binding protein Diffuse yjbN EGFP DY330 tRNA-dihydrouridine synthase A Diffuse yleA EGFP DY330 Hypothetical protein Diffuse (weak) yqcD EGFP DY330 Hypothetical protein Diffuse yqgE EGFP DY330 Hypothetical protein Diffuse yraO EGFP DY330 Hypothetical protein Diffuse ytfM EGFP DY330 Hypothetical protein Diffuse Diffuse Diffuse(weak) Diffuse Diffuse 1. The localization patterns of these proteins are observed under fluorescence microscope in the log or stationary phases of the corresponding E. coli strain. 2. # The localization results of these gene products are previous published by our lab (29). 53 Figure 5 Typical fluorescence microscopy images of live, immobilized E. coli DY330 cells containing a 3' EGFP coding sequence fused to a persistent gene on the chromosome. All panels are at the same magnification, and show DAPI fluorescent staining of (nucleoid) DNA (blue), EGFP fluorescence (green) and a merged image of the cells. From left to right and top to bottom, the images are of leuS-EGFP and glyA-EGFP which are distributed diffusely in the cell, groS-EGFP which forms a focus at one pole of the cell, rpsO-EGFP which forms foci at both poles of the cell, and atpG-EGFP and ccmF-EGFP which locate at the membrane. 54 Table 5 Localization patterns of EGFP-tagged ribosomal subunit proteins (rsp) ORF Description Subcelluar localization rplC 50S rspL3 Foci at both poles rplT 50S rspL20 Foci at both poles rplU 50S rspL21 Diffuse rplW 50S rspL23 Foci at both poles rpsO 30S rspS15 Foci at both poles rpsP 30S rspS16 Foci at both poles rpsQ 30S rspS17 Foci at both poles rpsU 30S rspS21 Foci at both poles 55 A B Figure 6 (A) Time-Lapse microscope image of E. coli with EGFP-tagged rpsO. Images were taken at 2 minute intervals, however, some time points were omitted due to there being no obvious change. (B) A schematic diagram of the change of the RpsO protein localization pattern during cell division. At the beginning, RpsO protein is located at the poles of the cell. When the cells start to divide, this protein can be found at the center of the cell as well as the poles of the cell. After division, RpsO protein is again located at the poles of the two cells. 56 3.3 Cell cycle and growth phase-specific protein localization patterns To test if the localization pattern will change with the growth of strain, a few successful EGFP tagged proteins were selected to observe the localization pattern at three different growth phases (lag phase, log phase, stationary phase) of DY330 strain. The samples collection time points correspond to these three growth phases are selected based on the growth curve of DY330 strain. Among these proteins, protein SuhB, generally annotated as inositol monophosphatase exhibited distinct localization patterns during different phases of cell growth. In the lag and stationary phases, the protein was found diffused throughout the cell cytoplasm, but it formed two focal points when the cells were in the log (exponential growth) phase (Figure7). But other proteins, like YhbC, show the same localization pattern at these three growth phases when are selected to observe (Figure 3). Considering the EGFP is fused to the C-terminus of target genes on chromosomes directly and the expression of EGFP is under the control of the target gene‟s promoter, the results can be viewed as the true expression pattern of target proteins. Due to the time limitation, not all tagged proteins in this project have been tested for their localization pattern change at different growth phase, but it is worth studying in the future. In addition to observing the change of localization pattern of EGFP-tagged proteins at different growth phases, the expression level of target proteins also can be tested. The advantage of applying recombineering technology to label target genes with fluorescent protein is the expression level of fluorescent protein that can represent the expression level of target proteins. Because the expression level of fluorescent protein gene can be determined by measuring the fluorescence intensity, the expression level of target genes can be represented by the fluorescence intensity. Several EGFP-tagged proteins have been selected to observe the fluorescence intensity at three different growth phases: lag phase, log phase and stationary phase. By comparing the fluorescence images of samples that are collected at different growth phases, it 57 is found that some proteins, show very different fluorescence intensity between different growth phases, even the fluorescence signal of several proteins is so weak that it is difficult to observe at log phase. But other proteins, like YhbC, show almost the same fluorescence intensity between different growth phases. This represents that there is no great change in expression level of these genes between these growth phases. 58 Figure 7 Change of localization pattern at different growth phases. All panels are at the same magnification and show DAPI fluorescent staining of (nucleoid) DNA (blue) and EGFP fluorescence (green). From top to bottom the phases are lag phase, log phase and stationary phase. (A) Fluorescence microscopy images of yhbC-EGFP E.coli showing the intensity of fluorescence signal and localization pattern are the same in the three growth phases. (B) Fluorescence microscopy images of suhB-EGFP E. coli showing differing localization patterns. In the lag phase and the stationary phase, the SuhB protein is found in the whole cell, but during log phase it forms foci in the cell. 59 3.4 ColiLo: an on-line catalogue of protein localization database for E. coli persistent proteins Our lab has created an on-line database: E. coli Protein Localization Database (named ColiLo) to store the results and associated data for the E. coli persistent protein localization experiments described here, and to facilitate its use by the scientific community. ColiLo allows users to search for genes through several ways: gene name, localization categories, function, and DNA or protein sequence. The webpage of every persistent gene whose tagged protein product could be successfully detected and visualized include two parts: the basic information of the gene, including synonym, function, description, DNA sequence, amino acid sequence and linkers to other related databases of gene and the localization related information, including all primers used for fluorescent proteinfluorescent protein tagging, the strain, localization category as well as representative fluorescence microscopy images of intracellular protein localization. For some selected proteins, experimental video is also uploaded. In the section of fluorescent microscopy images, a DAPI stain image and a fluorescence image are provided. A button named ' larger view ' can be found under each image and a corresponding separated larger image will pop up when click on it (Figure 8). ColiLo is open access to anyone who wants to find protein localization information and the main purpose of establishment of this database is to provide a research resource to the science community. The ColiLo database is accessible at: http://www.biochem.hku.hk/huanglab/Colilo/index.php. 60 A B Figure 8 The homepage and search result pages (GroS protein localization page as an example) of ColiLo database. In the webpage, searches can search by using a number of criteria, such as name of CDS/gene, subcellular location, protein function. Search will retrieve tagging primer sequence, description, images, videos that are used to assign protein localizations in this study. 61 Chapter 4: Discussion and Perspective 4.1 Organization of the proteome The intracellular localization patterns of the proteins encoded by the 611 persistent genes in E. coli have been studied (15 of them have been tagged and located previously in our lab by Watt et al (29)). DY330, a derivative of K12 sub-strain W3110, is used as our host organism because it contains a modified prophage region housing the thermally-inducible λ-Red system (PL promoter) under the tightly-regulated control of a temperature sensitive cI857 repressor. As such, it is possible to tag chromosomal genes using a highly-efficient recombineering approach, within a minimally-modified E. coli strain (143). 189 (~31%) of the 611 persistent genes within E. coli were successfully tagged at the C-terminus with EGFP, and the intracellular distributions of the encoded products of 139 of these (~23% of the total) were determined. Due to these successfully tagged proteins are persistent in E. coli, these C-terminal fusion proteins must retain at least a proportion of their native functionality. EGFP fusions of ca. 69% of the target genes could not be successfully created (Table 1). For 32% of the target genes, no colonies could be found on the plates of transformed cells; in the remainder, the EGFP-loxP-Cm-LoxP cassette was incorrectly positioned. A possible reason is that the EGFP protein might disturb the function of the target protein by disrupting its tertiary or quaternary structure (145) or the length of the linker between the persistent protein and EGFP fusion may not be optimal (146). Although EGFP is a widely used reporter in various organisms including E. coli due to its many advantages, the influence of EGFP to the correct folding of target proteins still cannot be excluded. The persistent genes are highly conserved within bacteria and are therefore likely to be essential under all or certain growth conditions (39). Their corresponding proteins would be expected to be strongly structurally conserved and structural perturbation might be more likely to result in lethality (39). As genes in E. coli are often organized into operons (147), insertion of a foreign DNA fragment within an operon could influence the transcription 62 and/or translation of downstream genes (i.e. polar effects) resulting in a lethal condition. There is also the possibility that fusions interrupted binding sequences for small regulatory RNA molecules. Fifty persistent genes were tagged successfully, but no fluorescent signal could be detected. This may be due to the fact that these genes were not expressed under the culture conditions used, or that the expression levels of the EGFP fusion proteins were just too low to be detected. In some of these strains, the selective marker (Chloramphenicol gene) was removed by “Cre-LoxP” system in order to check the reason for no signal. But there still no fluorescence signal can be observed after deleting the chloramphenicol gene. More strains should be tried to confirm if the selective marker is one of the reasons of no fluorescence signal in the future. Due to GFP that cannot be folded correctly in the periplasmic space (30), it is possible that the proteins located in the periplasmic space cannot be observed. One example is CcmE, as a membrane-anchor periplasmic heme chaperone, the C-terminal of CcmE is located in periplasm. No fluorescence signal can be detected in CcmE-EGFP strain (148). One possible solution for locating the CcmE protein is to label CcmE with EGFP at its N-terminus.The misfold of GFP when fused with other proteins also lead to failure of detecting fluorescent signal. A new engineered superfolder fluorescent protein can be employed as a possible solution (149). Related studies that sought to establish the localization of tagged proteins within microbial cells (on a genome-wide scale) have reported similar success rates. For example, researchers using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis were able to epitope-tag ca. 60% of the Saccharomyces cerevisiae proteome, enabling the subcellular locations of 45% of all yeast proteins to be determined (150). Given that the proteins tagged in this previous study were selected from the entire genome, and were not restricted to a subset of persistent genes of putative essentiality or importance, our success rate is reasonable. 63 4.2 Relationship of protein localization and function The protein localizations identified here were, by and large, consistent with known protein functions. For example, the AtpG protein found located on the membrane in this projectis consistent with its function as a component of membrane-bound ATP synthase. Another typical example is RecA protein. As a DNA repair protein, it should form foci at any position on the chromosome (151). The localization result of RecA on foci of the RecA-EGFP protein were found co-incident with nucleoid DNA during different growth phases and at different points in the cell cycle which is expected according to its function (Figure 9). Figure 9 The localization pattern of the EGFP tagged recA gene product. DAPI fluorescent staining of (nucleoid) DNA (blue) and EGFP fluorescence (green) are shown. The recA gene product forms foci at a pole of the cell or at the center of the cell (Left fluorescence image) or near the pole of the cell (Right fluorescence image). 64 CcmB, CcmC and CcmE are three of the proteins are involved in the maturation of c-type cytochrome in Gram-negative bacteria (152). Each of these three proteins play different roles in the maturation of c-type cytochrome and this determine the different subcellular localization between them. As membrane-integral component of ABC-transporter, CcmB and CcmC both have six membrane helices. The N- and C- terminal of them are in the cytoplasm. Consistent with their function, both these two proteins are located on the membrane in this project. But to CcmE, it is completely different. The function of CcmE that binds heme delivered by CcmC and transfers it to apo-cytochromes determines CcmE is a periplasmic protein that has its N-terminus attached to the membrane and C-terminus is in the periplasm. This is reflected in this project that it is failed in determining the subcellular localization of CcmE protein by labeling it with EGFP at its C-terminus because EGFP protein cannot be folded correctly in the periplasm. All the data strongly provide support for the close relationship between protein localization and function. On the basis of the fact that protein localization is closely related to its function, proteins showing different localization pattern in this project may imply other unknown functions. Several ribosome subunit proteins were found to form foci at the poles of the cell rather than to distribute diffusely in the cell as previously reported (27). Previous works show that RpsO, one such protein, is not essential for ribosome formation and cell growth at 37oC. However, at 25 oC, it appears to be essential for viability, and a ribosome biogenesis defect is apparent under those conditions if RpsO is deleted (153). Besides its role as a ribosomal protein, RpsO is also involved in transcription termination and mRNA turnover and in particular in the control of RNase III activity (154). It is required for the optimal synthesis of lipoproteins and mutations in RpsO can selectively affect the synthesis of exported proteins such as constitutively expressed lipoproteins and inducible MBP, albeit to different extents, but it has little effect on the synthesis of OmpA (155). The results here (Figure 4) indicate that the localization of RpsO varies during cell division, suggesting either an additional role in this process or that specific steps of RNA degradation are 65 compartmentalized in the cell (156, 157). It is reported that some proteins show the change of localization pattern at different growth temperature in E. coli (158). The localization pattern of RpsO at three different growth temperatures (18℃, 32℃, 42℃) also be observed, but there is no obvious localization pattern change between different temperature (Figure 10). Combined with the experiments of last section, it can draw a conclusion that the subcellular localization RpsO, one of ribosomal subunit proteins, is dynamic in the process of cell division but it is not changed by growth phase and culture temperature. But in Bacilllus subtilis, Mascarenhas et al found that ribosomal protein L1 localized around the nucleoids, preponderantly close to the cell pole, in growing cell but distribute throughout cell in stationary phase by tagging this protein with blue fluorescent protein (BFP) (159). 66 18℃ 32℃ 42℃ Figure 10 The localization pattern of RpsO-EGFP at different growth temperatures. From left to right: 18℃, 32℃, 42℃. At all these temperatures, RpsO protein forms two foci at the each end of cell. 67 In addition to playing an important role in translation, a number of ribosomal proteins also have additional functions. For example, the ribosomal protein S16 (RpsP), an essential component of the 30S ribosomal particle, exhibits a Mg2+/Mn2+-dependent endonuclease activity (160). In eukaryotes, the RpS3 ribosomal protein is involved in DNA repair (161-163). Proteins associated with cell division, repair and partitioning of the chromosome often form dynamic, polar or midcell localization patterns (129, 151, 164, 165). It may be worthwhile, based on the localization patterns reported here, to examine other ribosomal proteins for these functions.Mascarenhas et al found that ribosomal protein L1 of Bacilllus subtilis localized around the nucleoids, preponderantly close to the cell pole, in growing cell by tagging this protein with blue fluorescent protein (BFP). Our results confirm that polar and asymmetrical intracellular protein distributions are not uncommon in E. coli. For example, several proteins (e.g RpsO, RpsU) were predominantly located at both cell poles, indicating that this distribution may relate to a currently unknown function of these proteins. However, misfolded proteins may be deposited at the cell poles due to aggregation, as evidenced by their association with disaggregating chaperones (158). If this is the case, tagged protein libraries could provide a way to study the folding and unfolding of the fusion proteins under different conditions. Due to the complexity of protein interaction networks, protein interactions also might influence a protein‟s localization pattern (29, 92). 4.3 The dynamic of protein localization In general, the dynamic of protein localization happen on those proteins that form foci in the cell and is related to the special function of these proteins. Bejerano-Sagie et al reported that DisA, a nonspecific DNA binding protein of Bacillus subtilis, forms a single focus (166). The time-lapse experiment demonstrates that this focus move in the bacterial cell and it will stop at sites of DNA damage. It is also reported that this movement is chromosome integrity and energy but not DNA dependent. Another reported dynamic pattern is PopZ protein of Caulobacter crescentus. PopZ, bind to the ParB protein, remains 68 located at the pole of the swarmer cell upon the cell transfer to the stalked cell. In the stalked cell, PopZ form foci at two poles of the cell until cell division (144). Unlike DisA protein which moves around the cell and PopZ protein stays at one pole of the cell, RpsO protein is found to have a fully different dynamic pattern in this project. RpsO, which encodes the 30S ribosomal subunit protein S15, form two foci at poles of the cell. When the cell begins to divide, a focus can be found at the centre of the cell. After the division is finished, there are two foci that can be found at the poles of the each of the two new cells again. Although it is failed to observe that RpsO forms a ring structure at the mid-point of cell, its dynamic pattern is more like that of cell division proteins, FtsZ, which forms ring at the mid-cell division site. Another dynamic of protein localization is the change of the localization pattern at the different growth phases of E. coli. A number of proteins, such as SuhB, showed distinct localization patterns during different growth phases (Fig 5B), indicating that the growth phase of an organism should be taken into consideration when determining protein localization patterns. SuhB is also a protein involved in RNA metabolism, with a function that has not yet been clearly established, and is most probably not only that of an inositol phosphatase. In this respect it may be of interest to notice that SuhB is similar to phosphatase CysQ, specific for degradation of 3', 5‟-adenosine bisphosphate suggesting a role in phosphorylated nucleosides metabolism. Fluorescence intensity levels varied among the different EGFP-tagged proteins, and sometimes for the same protein during different growth phases. For example, tRNA(Ile)-lysidine synthetase (TilS, YaeN), showed different fluorescence intensity levels in the exponential and stationary phases: weak fluorescence signal can be observed in the stationary phase, but it was hardly observed in the exponential phase. This correlates well with the variation of abundance of tRNA (Ile) AUA during growth (167, 168).. One advantage of chromosome-based gene tagging is that the reporter protein signal is much more likely to accurately reflect the native protein expression levels than a plasmid-based expression system, since the fusion protein gene is a single copy 69 and under the control of the native promoter. This makes it possible to use the fluorescence intensity to monitor and quantify the target proteins‟ expression levels (93). 4.4 Conclusions and perspective This work has provided detailed information on the intracellular localization of the protein products in 23% of the persistent genes in E. coli. It has shown that the growth phase of the cell must be considered in protein localization studies. Some of the ribosomal proteins may have functions in addition to those related to protein biogenesis, based on their differences in intracellular distributions. The techniques in this work have also resulted in tools and methods that could be used for studies on protein folding and gene expression. These experimental data and images are freely accessible and searchable at the ColiLo database. The tagged E. coli strains are freely available to the scientific community upon requested. With more and more knowledge about the organism, we are not only focus on disclosing the life secret but become interested in design and construction of new biological systems. To construct a new biological system, the most important is making the persistent genes expression at the appropriate time and the appropriate cell compartment. It is understandably easy to image what will happen if a membrane anchored protein is located to cytoplasm when we designing a new biology system. In order to make proteins, especially those proteins that essential to cell survival, perform their normal functions in a new biology system, the correct knowledge of protein localization is necessary. This project is the first report of the genome-scale protein localization by labeling proteins on chromosome directly in E. coli as far as I know and the research source provided in this project can give useful information on it: one hand, some previous protein subcelluar localization results of E.coli were further confirmed by labeling proteins on chromosome directly; On the other hand, some new protein subcelluar localization results also be reported. By combining our protein localization source with other researcher‟s results, the 70 more accurate protein localization map of E. coli can be described and it will enable us to understand the protein function and metabolism of E. coli better. It has been proven in this project that using recombineering method to tag the coding region of E. coli on chromosome directly can give us some fresh insight on the function and localization of proteins although not all genes can be tagged successfully at their C-terminus. In order to tag all persistent gene products of E. coli successfully, refer to other localization research works, and the N-terminal EGFP tagging would be tried in the future. Second, due to some proteins showing different localization pattern from previous results, like some ribosome subunit proteins, it may represent new function of these proteins, of which the mechanism is worthy of further research. In my study, it is found that different proteins show different fluorescence intensity. Considered with the advantage of recombineering technology that the expression of EGFP is under the control of the promoter of target gene and it means the expression level of EGFP represents the expression level of labeled protein, the recombineering technology can be used as tool of quantitative analysis of gene expression (93). Quantitative analysis plays important roles in understanding the celluar behavior. The key point in quantitative analysis is connecting the fluorescence intensity with the copy number of protein. Although several algorithm methods have been reported (169,170), more accurate methods that allow unbiased estimation of copy number of protein still need to further study.On the other hand, by measuring the fluorescence intensity, it can be used as an indicator of the change of expression level of target genes caused by various factors. For example, the intensity of fluorescence can be a biosensor of the change of corresponding environment factors when the expression of one gene varies with these factors; if the expression level variation of genes are influenced by the expression fluctuation of other gene(s), it is useful in the study of metabolism regulate network. This successfully constructed C-terminal EGFP-tagging E. coli persistent gene library and recombineering technology also provides a platform to study the strength of promoter by quantifying the fluorescence intensity. Now there are 71 several different color fluorescent proteins, like ECFP 、 EYFP、mRFP, different proteins can be co-labeled different fluorescent proteins in a cell by recombineering technology to study the interaction between them. Recombineering has been widely used in E. coli, from protein localization research by labeling reporter to determine protein interactions by tagging different reporters (91). 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(2009) Recombineering: a homologous recombination-based method of genetic engineering, Nature Protocol 4, 206-223. 98 Appendix 1 The fluorescence images of successfully tagged E. coli persistent genes CDS abC Description/Function D-methionine transport ATP-binding protein Acetyl-coenzyme accD aceF A carboxylase carboxyltransferase subunit beta Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex 99 Localization pattern CDS apaH aroB aroC Description/Function Bis(5'-nucleosyl)-tetraphosphatase 3-dehydroquinate synthase Chorismate synthase F1 sector of membrane-bound atpG ATP synthase Gamma subunit bioA 7,8-diaminopelargonic acid synthase PLP-dependent 100 Localization pattern CDS bioB bioF Description/Function Biotin synthase 8-amino-7-oxononanoate synthase Carbamoyl phosphate synthetase small subunit carA 101 Localization pattern CDS Description/Function Carbamoyl-phosphate carB synthase large subunit ccmA Cytochrome c biogenesis ATP-binding export protein ccmB Heme exporter protein B ccmC Heme exporter protein C ccmF Cytochrome c-type biogenesis protein 102 Localization pattern CDS clpB Description/Function Protein disaggregation chaperone Conserved hypothetical protein crcB cysS Putative inner membrane protein associated with chromosome condensation Cysteinyl-tRNA synthetase 103 Localization pattern CDS Description/Function deF Peptide deformylase dnaE DNA polymerase III alpha subunit dsbB Disulfide bond formation protein B 104 Localization pattern CDS Description/Function efP Elongation factor EF-P enO Enolase folB folD Dihydroneopterin aldolase Bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase and 5,10-methylene-tetrahydrofolate cyclohydrolase 105 Localization pattern CDS Description/Function folK 2-amino-4-hydroxy-6-hydroxymethy ldihydropteridine pyrophosphokinase frR Ribosome recycling factor fuR Ferric uptake regulation protein Methyltransferase gidB 106 Localization pattern CDS Description/Function glnS Glutaminyl-tRNA synthetase glyA Serine hydroxymethyltransferase groS Cpn10 chaperonin GroES Small subunit of GroESL hemE Uroporphyrinogen decarboxylase 107 Localization pattern CDS hemH Description/Function Ferrochelatase hflK HflA complex cleaves lambda cII hisA N-(5'-phospho-L-ribosyl-formimino) -5-amino-1-(5'phosphoribosyl)-4-imidazolecarboxa mide isomerase hslU ATP-dependent hsl protease ATP-binding subunit 108 Localization pattern CDS Description/Function hslV ATP-dependent protease htpG Chaperone hupB DNA-binding protein HU-beta ileS Isoleucyl-tRNA synthetase 109 Localization pattern CDS Description/Function infB Translation initiation factor IF-2 kdsB 3-deoxy-manno-octulosonate cytidylyltransferase ksgA Dimethyladenosine transferase leuS Leucyl-tRNA synthetase 110 Localization pattern CDS Description/Function loN ATP-dependent protease lpxA Acyl-[acyl-carrier-protein]--UDP-N-a cetylglucosamine O-acyltransferase lpxC UDP-3-O-acyl N-acetylglucosamine deacetylase 111 Localization pattern CDS Description/Function 5,10-methylenetetrahydrofolate metF reductase miaA Delta(2)-isopentenylpyrophosphate tRNA-adenosine transferase UDP-N-acetylmuramate mpL L-alanyl-gamma-D-glutamyl-me so-diaminopimelate ligase 112 Localization pattern CDS Description/Function Cell wall structural complex mreC MreBCD transmembrane component MreC murB UDP-N-acetylenolpyruvoylglucosami ne reductase 113 Localization pattern CDS Description/Function pepA Aminopeptidase A pepN Aminopeptidase N pgK Phosphoglycerate kinase 114 Localization pattern CDS Description/Function ppA Inorganic pyrophosphatase prfC Peptide chain release factor RF-3 prlC Oligopeptidase A 115 Localization pattern CDS Description/Function prmA Ribosomal protein L11 methyltransferase proA Gamma-glutamylphosphate reductase proC Pyrroline-5-carboxylate reductase psD phosphatidylserine decarboxylase proenzyme 116 Localization pattern CDS Description/Function ptH Peptidyl-tRNA hydrolase pykA queA recA Pyruvate kinase II S-adenosylmethionine:tRNA ribosyltransferase-isomerase General recombination and DNA repair protein 117 Localization pattern CDS Description/Function recJ Single-stranded-DNA-specific exonuclease rhO Transcription termination factor ribC Riboflavin synthase alpha subunit 118 Localization pattern CDS Description/Function ribD Riboflavin biosynthesis protein rluD Ribosomal large subunit pseudouridine synthase D rnE Ribonuclease E rnhA Ribonuclease HI 119 Localization pattern CDS rnR Description/Function Ribonuclease R rpE Ribulose-phosphate 3-epimerase rpH Ribonuclease PH rpiA Ribose-5-phosphate isomerase A 120 Localization pattern CDS rplC Description/Function 50S ribosomal subunit protein L3 50S ribosomal subunit protein L20 rplT 121 Localization pattern CDS Description/Function 50S ribosomal subunit protein L21 rplU rplW 50S ribosomal subunit protein L23 rpoH RNA polymerase sigma-32 factor DNA-directed RNA polymerase rpoZ omega chain 122 Localization pattern CDS Description/Function rpsO 30S ribosomal subunit protein S15 rpsP 30S ribosomal subunit protein S16 rpsQ 30S ribosomal subunit protein S17 123 Localization pattern CDS Description/Function rpsU 30S ribosomal subunit protein S21 rsmB Ribosomal RNA small subunit methyltransferase B secB serA Protein-export protein D-3-phosphoglycerate dehydrogenase 124 Localization pattern CDS serC serS Description/Function Localization pattern Phosphoserine aminotransferase Seryl-tRNA synthetase \ smF Smf protein smpB SsrA-binding protein 125 CDS Description/Function Guanosine-3',5'-bis(diphosphate) spoT 3'-pyrophosphohydrolase sspA Stringent starvation protein A suhB Inositol-1-monophosphatase talB Transaldolase B 126 Localization pattern CDS Description/Function tktA Transketolase 1 tpiA Triosephosphate isomerase Tryptophan synthase trpA alpha chain trxA Thioredoxin 1 127 Localization pattern CDS tufB usG yaeN Description/Function Protein chain elongation factor EF-Tu Stabilizes phage lambda protein N-NusA-RNAP antitermination comple Putative cell cycle protein mesJ 128 Localization pattern CDS Description/Function ybeB Hypothetical protein ydaO Hypothetical protein ydhH Hypothetical protein 129 Localization pattern CDS Description/Function yfgB Hypothetical protein yfhQ Hypothetical tRNA/rRNA methyltransferase 130 Localization pattern CDS ygcA Description/Function 23S rRNA (Uracil-5-)-methyltransferase ygfB Hypothetical protein yggW Hypothetical protein yhbC Hypothetical protein 131 Localization pattern CDS Description/Function yhbY Putative RNA-binding protein yhbZ Putative GTP-binding factor yhdG tRNA-dihydrouridine synthase B 132 Localization pattern CDS Description/Function yihA GTP-binding protein yjbN tRNA-dihydrouridine synthase A yleA Hypothetical protein 133 Localization pattern CDS Description/Function yqcD Hypothetical protein yqgE Hypothetical protein yraO ytfM 1. Localization pattern Hypothetical protein Hypothetical protein The localization patterns of these proteins are observed under fluorescence microscope in the log phase or stationary phase of the corresponding EGFP tagged DY330 E.coli strain cultured at 32°C. 134 Appendix 2 The list of all 611persistent genes of E. coli Gene Category of tagging result Essentiality* Short Description aroK 1 N Shikimate kinase I aspS 1 E Aspartyl-tRNA synthetase atpC 1 N ATP synthase epsilon chain bcp 1 N Bacterioferritin comigratory protein bolA 1 N Morphogene cafA 1 N Ribonuclease G ccmE 1 N Cytochrome c-type biogenesis protein ccmG 1 N Thiol:disulfide interchange protein cdsA 1 E Phosphatidate cytidylyltransferase cydA 1 N Cytochrome d ubiquinol oxidase subunit I cysK 1 N Cysteine synthase A cysQ 1 N PAP (3',5' adenosine diphosphate) 3' phosphatase dfp 1 E Coenzyme A biosynthesis bifunctional protein dnaA 1 E Chromosomal replication initiator protein dut 1 E Deoxyuridine 5'-triphosphate nucleotidohydrolase dxr 1 E 1-deoxy-D-xylulose 5-phosphate reductoisomerase fabD 1 E Malonyl CoA-acyl carrier protein transacylase fabH 1 N 3-oxoacyl-[acyl-carrier-protein] synthase III fadD 1 N Long-chain-fatty-acid--CoA ligase 135 folC 1 N FolC bifunctional protein folP 1 N Dihydropteroate synthase ftsI 1 E Peptidoglycan synthetase ftsI ftsJ 1 E Ribosomal RNA large subunit methyltransferase J ftsX 1 E Cell division protein gcvH 1 N Glycine cleavage system H protein glmS 1 N Glucosamine--fructose-6-phosphate aminotransferase [isomerizing] glnD 1 N [Protein-PII] uridylyltransferase glnE 1 N Glutamate-ammonia-ligase adenylyltransferase glpK 1 N Glycerol kinase gltX 1 E Glutamyl-tRNA synthetase glyQ 1 E Glycyl-tRNA synthetase alpha chain glyS 1 E Glycyl-tRNA synthetase beta chain gmk 1 E Guanylate kinase gor 1 N Glutathione reductase gph 1 N Phosphoglycolate phosphatase gpsA 1 N Glycerol-3-phosphate dehydrogenase [NAD(P)+] gyrA 1 E DNA gyrase subunit A gyrB 1 E DNA gyrase subunit B hemA 1 N Glutamyl-tRNA reductase hemB 1 N Delta-aminolevulinic acid dehydratase hemF 1 N Coproporphyrinogen III oxidase, aerobic 136 hemL 1 N Glutamate-1-semialdehyde 2,1-aminomutase hflB 1 E Cell division protein hfq 1 N HF-I, host factor for phage Qbeta hisF 1 N Imidazole glycerol phosphate synthase subunit holA 1 E DNA polymerase III, delta subunit holB 1 E DNA polymerase III, delta' subunit hrpA 1 N ATP-dependent helicase ihfA 1 N Integration host factor alpha-subunit ihfB 1 N Integration host factor beta-subunit ilvD 1 N Dihydroxy-acid dehydratase lepB 1 E Signal peptidase I ligA 1 E DNA ligase lipB 1 N Lipoyltransferase mazG 1 N Nucleoside triphosphate pyrophosphohydrolase mrp 1 N Putative ATPase msbA 1 E Lipid A export ATP-binding/permease protein murE 1 E UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase murF 1 E UDP-N-acetylmuramoyl-tripeptide--D-alanyl-D-alanine ligase mutL 1 N DNA mismatch repair protein mutM 1 N Formamidopyrimidine-DNA glycosylase mutY 1 N A/G-specific adenine glycosylase ndk 1 N Nucleoside diphosphate kinase 137 ogt 1 N Methylated-DNA--protein-cysteine methyltransferase orn 1 U Oligoribonuclease oxyR 1 N Hydrogen peroxide-inducible genes activator panB 1 N 3-methyl-2-oxobutanoate hydroxymethyltransferase parC 1 E Topoisomerase IV subunit A pgsA 1 E CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase pmbA 1 N Antibiotic peptide MccB17 pntB 1 N NAD(P) transhydrogenase subunit beta polA 1 E DNA polymerase I prfA 1 E Peptide chain release factor 1 proB 1 N Glutamate 5-kinase proS 1 E Prolyl-tRNA synthetase purA 1 N Adenylosuccinate synthetase purD 1 N Phosphoribosylamine--glycine ligase purE 1 N Phosphoribosylaminoimidazole carboxylase catalytic subunit purF 1 N Amidophosphoribosyltransferase purK 1 N Phosphoribosylaminoimidazole carboxylase ATPase subunit purL 1 N Phosphoribosylformylglycinamidine synthase purM 1 N Phosphoribosylformylglycinamidine cyclo-ligase purN 1 N Phosphoribosylglycinamide formyltransferase pyrD 1 N Dihydroorotate dehydrogenase pyrE 1 N Orotate phosphoribosyltransferase 138 radA 1 N DNA repair protein recD 1 N Exodeoxyribonuclease V alpha chain rep 1 N ATP-dependent DNA helicase rimI 1 N Ribosomal-protein-alanine acetyltransferase rimM 1 N 16S rRNA processing protein rimM rluC 1 U Ribosomal large subunit pseudouridine synthase C rnc 1 N Ribonuclease III rnpA 1 E Ribonuclease P protein component rplB 1 E 50S ribosomal protein L2 rplD 1 E 50S ribosomal protein L4 rplF 1 E 50S ribosomal protein L6 rplR 1 E 50S ribosomal protein L18 rplI 1 E 50S ribosomal protein L9 rplJ 1 E 50S ribosomal protein L10 rplM 1 E 50S ribosomal protein L13 rplN 1 E 50S ribosomal protein L14 rplP 1 E 50S ribosomal protein L16 rplS 1 E 50S ribosomal protein L19 rpmA 1 E 50S ribosomal protein L27 rpmB 1 E 50S ribosomal protein L28 rpmG 1 E 50S ribosomal protein L33 rpmH 1 E 50S ribosomal protein L34 139 rpmI 1 E 50S ribosomal protein L35 rpoB 1 E DNA-directed RNA polymerase beta chain rpoE 1 N RNA polymerase sigma-E factor rpsA 1 E 30S ribosomal protein S1 rpsB 1 E 30S ribosomal protein S2 rpsC 1 E 30S ribosomal protein S3 rpsE 1 E 30S ribosomal protein S5 rpsF 1 E 30S ribosomal protein S6 rpsI 1 E 30S ribosomal protein S9 rpsJ 1 E 30S ribosomal protein S10 rpsK 1 E 30S ribosomal protein S11 rpsL 1 E 30S ribosomal protein S12 rpsM 1 E 30S ribosomal protein S13 rpsN 1 E 30S ribosomal protein S14 rpsR 1 E 30S ribosomal protein S18 rpsS 1 E 30S ribosomal protein S19 rpsT 1 N 30S ribosomal protein S20 secD 1 N Protein-export membrane protein secE 1 E Preprotein translocase secE subunit secG 1 N Protein-export membrane protein slyD 1 N FKBP-type peptidyl-prolyl cis-trans isomerase slyD sspB 1 N Stringent starvation protein B 140 sucA 1 N 2-oxoglutarate dehydrogenase E1 component sucB 1 N Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex sucD 1 N Succinyl-CoA synthetase alpha chain tag 1 N DNA-3-methyladenine glycosylase I tig 1 N Trigger factor tldD 1 U TldD protein tolB 1 N TolB protein tolC 1 N Specific tolerance to ColE1; affects chromosome segregation; OM porin topA 1 E DNA topoisomerase I trmD 1 E tRNA (Guanine-N(1)-)-methyltransferase trpE 1 N Anthranilate synthase component I trpS 1 E Tryptophanyl-tRNA synthetase trxB 1 N Thioredoxin reductase ung 1 N Uracil-DNA glycosylase uup 1 U ABC transporter ATP-binding protein uvrB 1 N UvrABC system protein B uvrC 1 N UvrABC system protein C valS 1 E Valyl-tRNA synthetase xerC 1 N Tyrosine recombinase xerD 1 N Tyrosine recombinase xthA 1 N Exodeoxyribonuclease III yadG 1 N Hypothetical ABC transporter ATP-binding protein 141 yaeC 1 U D-methionine-binding lipoprotein yajQ 1 U Hypothetical protein ybaB 1 U Hypothetical protein ybaD 1 U Hypothetical protein ybaX 1 U Hypothetical protein ybeD 1 U Hypothetical protein ybeY 1 U Hypothetical protein ycaJ 1 U Hypothetical protein ycfF 1 U HIT-like protein yciB 1 N Probable intracellular septation protein ydgM 1 N Electron transport complex protein yeaZ 1 U Hypothetical M22 peptidase homolog yffB 1 U Hypothetical protein yfhF 1 U Hypothetical protein yfhL 1 U Putative ferredoxin-like protein yfhP 1 U Hypothetical protein yfiH 1 U Hypothetical protein yfjF 1 U Hypothetical protein yfjG 1 U Hypothetical protein ygfA 1 U Hypothetical protein ygfE 1 N Hypothetical protein yggS 1 U Hypothetical protein 142 yggT 1 U Hypothetical protein yggV 1 U HAM1 protein homolog yggX 1 U Hypothetical protein yhbN 1 U Putative transport protein (ABC superfamily) yhgH 1 N Hypothetical protein yhgI 1 N Protein gntY yhiR 1 U Hypothetical protein yibK 1 U Hypothetical tRNA/rRNA methyltransferase yibN 1 U Hypothetical protein yibP 1 U Hypothetical protein yidC 1 E Inner membrane protein yjeA 1 U Putative lysyl-tRNA synthetase yjeE 1 E Hypothetical protein yjeK 1 U Hypothetical protein yjjV 1 U Putative deoxyribonuclease yraL 1 U Hypothetical protein yrbD 1 U Hypothetical protein yrbE 1 U Hypothetical protein yrdC 1 U Hypothetical protein yrfE 1 U ADP compounds hydrolase yrfI 1 U 33 kDa chaperonin abc 2 U D-methionine transport ATP-binding protein 143 accD 2 E Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta aceF 2 N Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex adk * 2 E Adenylate kinase apaH 2 N Bis(5'-nucleosyl)-tetraphosphatase, symmetrical aroB 2 N 3-dehydroquinate synthase aroC 2 N Chorismate synthase atpG 2 N ATP synthase gamma chain bioA 2 N Adenosylmethionine-8-amino-7-oxononanoate aminotransferase bioB 2 N Biotin synthase bioF 2 N 8-amino-7-oxononanoate synthase carA 2 N Carbamoyl-phosphate synthase small chain carB 2 N Carbamoyl-phosphate synthase large chain ccmA 2 N Cytochrome c biogenesis ATP-binding export protein ccmB 2 N Heme exporter protein B ccmC 2 N Heme exporter protein C ccmF 2 N Cytochrome c-type biogenesis protein clpB 2 N Chaperone crcB 2 U Protein crcB cysS 2 E Cysteinyl-tRNA synthetase def 2 E Peptide deformylase dnaE 2 E DNA polymerase III alpha subunit dsbB 2 N Disulfide bond formation protein B 144 dxS* 2 E 1-deoxy-xylulose phosphate synthase efp 2 E Elongation factor P eno 2 E Enolase erA* 2 E Ras-like GTP-binding protein folB 2 E Dihydroneopterin aldolase folD 2 N FolD bifunctional protein folK 2 N 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase frr 2 E Ribosome recycling factor fur 2 N Ferric uptake regulation protein gidB 2 N Methyltransferase glnS 2 E Glutaminyl-tRNA synthetase glyA 2 N Serine hydroxymethyltransferase groS 2 E 10 kDa chaperonin hemE 2 N Uroporphyrinogen decarboxylase hemH 2 N Ferrochelatase hflK 2 N HflA complex cleaves lambda cII hisA 2 N 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase hslU 2 N ATP-dependent hsl protease ATP-binding subunit hslV 2 N ATP-dependent protease htpG 2 N Chaperone hupB 2 N DNA-binding protein HU-beta ileS 2 E Isoleucyl-tRNA synthetase 145 infB 2 E Translation initiation factor IF-2 kdsB 2 E 3-deoxy-manno-octulosonate cytidylyltransferase ksgA 2 N Dimethyladenosine transferase leuS 2 E Leucyl-tRNA synthetase lon 2 N ATP-dependent protease lpxA 2 E Acyl-[acyl-carrier-protein]--UDP-N-acetylglucosamine O-acyltransferase lpxC 2 E UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase map* 2 E Methionine amino peptidase metF 2 N 5,10-methylenetetrahydrofolate reductase metK* 2 E S-adenosyl-methionine synthetase miaA 2 N tRNA delta(2)-isopentenylpyrophosphate transferase mpl 2 N UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase mreC 2 N Rod shape-determining protein murB 2 E UDP-N-acetylenolpyruvoylglucosamine reductase nusA* 2 E Transcription elongation protein pepA 2 N Cytosol aminopeptidase pepN 2 N Aminopeptidase N pgk 2 N Phosphoglycerate kinase ppa 2 E Inorganic pyrophosphatase prfC 2 N Peptide chain release factor 3 prlC 2 N Oligopeptidase A prmA 2 N Ribosomal protein L11 methyltransferase 146 proA 2 N Gamma-glutamyl phosphate reductase proC 2 N Pyrroline-5-carboxylate reductase psd 2 N Phosphatidylserine decarboxylase proenzyme pth 2 E Peptidyl-tRNA hydrolase pykA 2 N Pyruvate kinase II pyrH* 2 E Uridylate kinase queA 2 N S-adenosylmethionine:tRNA ribosyltransferase-isomerase recA 2 N General recombination and DNA repair protein recG* 2 N ATP-dependent DNA helicase recJ 2 N Single-stranded-DNA-specific exonuclease rho 2 E Transcription termination factor ribC 2 N Riboflavin synthase alpha chain ribD 2 N Riboflavin biosynthesis protein rluD 2 N Ribosomal large subunit pseudouridine synthase D rne 2 E Ribonuclease E rnhA 2 N Ribonuclease HI rnr 2 N Ribonuclease R rpe 2 N Ribulose-phosphate 3-epimerase rph 2 N Ribonuclease PH rpiA 2 N Ribose-5-phosphate isomerase A rplC 2 E 50S ribosomal protein L3 rplT 2 E 50S ribosomal protein L20 147 rplU 2 E 50S ribosomal protein L21 rplW 2 E 50S ribosomal protein L23 rpoH 2 N RNA polymerase sigma-32 factor rpoZ 2 N DNA-directed RNA polymerase omega chain rpsO 2 E 30S ribosomal protein S15 rpsP 2 E 30S ribosomal protein S16 rpsQ 2 E 30S ribosomal protein S17 rpsU 2 E 30S ribosomal protein S21 rsmB 2 N Ribosomal RNA small subunit methyltransferase B secB 2 N Protein-export protein serA 2 N D-3-phosphoglycerate dehydrogenase serC 2 N Phosphoserine aminotransferase serS 2 E Seryl-tRNA synthetase smf 2 N Smf protein smpB 2 N SsrA-binding protein spoT 2 E Guanosine-3',5'-bis(diphosphate) 3'-pyrophosphohydrolase sspA 2 N Stringent starvation protein A suhB 2 N Inositol-1-monophosphatase talB 2 N Transaldolase B tktA 2 N Transketolase 1 tpiA 2 N Triosephosphate isomerase trmE* 2 N Probable tRNA modification GTPase 148 trpA 2 N Tryptophan synthase alpha chain trxA 2 N Thioredoxin 1 tufB 2 N Elongation factor Tu usg 2 E Stabilizes phage lambda protein N-NusA-RNAP antitermination complex yaeL* 2 E Inner membrane zinc metalloprotease ecfE yaeN 2 E Putative cell cycle protein mesJ ybeB 2 U Hypothetical protein ydaO 2 N Hypothetical protein ydhH 2 U Hypothetical protein yfgB 2 U Hypothetical protein yfgK* 2 U GTP-binding protein yfhQ 2 U Hypothetical tRNA/rRNA methyltransferase yfjB* 2 U Probable inorganic polyphosphate/ATP-NAD kinase ygcA 2 N 23S rRNA (Uracil-5-)-methyltransferase ygfB 2 U Hypothetical protein yggW 2 U Hypothetical protein ygjD* 2 E Probable O-sialoglycoprotein endopeptidase yhbC 2 U Hypothetical protein yhbY 2 U Putative RNA binding protein yhbZ 2 E Hypothetical GTP-binding protein yhdG 2 U tRNA-dihydrouridine synthase B yihA 2 E Probable GTP-binding protein 149 yjbN 2 U tRNA-dihydrouridine synthase A yleA 2 U Hypothetical protein yqcD 2 N Hypothetical protein yqgE 2 U Hypothetical protein yraO 2 U Hypothetical protein ytfM 2 U Hypothetical protein aroE 3 N Shikimate dehydrogenase atpB 3 N ATP synthase a chain atpE 3 N ATP synthase C chain atpF 3 N ATP synthase B chain bcr 3 N Bicyclomycin resistance protein clpP 3 N ATP-dependent Clp protease proteolytic subunit cmk 3 N Cytidylate kinase csrA 3 N Carbon storage regulator cydB 3 N Cytochrome d ubiquinol oxidase subunit II dapB 3 N Dihydrodipicolinate reductase dapF 3 N Diaminopimelate epimerase dksA 3 N DnaK suppressor protein dnaN 3 E DNA polymerase III, beta chain dsbC 3 N Thiol:disulfide interchange protein eda 3 N KHG/KDPG aldolase fmt 3 E Methionyl-tRNA formyltransferase 150 folE 3 N GTP cyclohydrolase I gltA 3 N Citrate synthase guaA 3 N GMP synthase [glutamine-hydrolyzing] infA 3 E Translation initiation factor IF-1 kdtA 3 E 3-deoxy-D-manno-octulosonic-acid transferase lepA 3 N GTP-binding protein lipA 3 N Lipoyl synthase lpxK 3 U Tetraacyldisaccharide 4'-kinase mrdA 3 E Penicillin-binding protein 2 murD 3 E UDP-N-acetylmuramoylalanine--D-glutamate ligase parE 3 E Topoisomerase IV subunit B pcnB 3 N Poly(A) polymerase pgi 3 N Glucose-6-phosphate isomerase phoB 3 N Phosphate regulon transcriptional regulatory protein ppdD 3 N Prepilin peptidase dependent protein D recF 3 N DNA replication and repair protein relA 3 N GTP pyrophosphokinase rnhB 3 N Ribonuclease HII rpmF 3 E 50S ribosomal protein L32 ruvB 3 N Holliday junction DNA helicase ruvC 3 N Crossover junction endodeoxyribonuclease sdhA 3 N Succinate dehydrogenase flavoprotein subunit 151 sdhB 3 N Succinate dehydrogenase iron-sulfur protein tgt 3 N Queuine tRNA-ribosyltransferase ubiE 3 U Ubiquinone/menaquinone biosynthesis methyltransferase upp 3 N Uracil phosphoribosyltransferase ybeZ 3 U PhoH-like protein ydhD 3 U Probable monothiol glutaredoxin ydjA 3 N Hypothetical protein yfcH 3 N Hypothetical protein yggH 3 U tRNA (guanine-N(7)-)-methyltransferase yggJ 3 U Hypothetical protein yggR 3 U Hypothetical protein yrbF 3 U Hypothetical ABC transporter ATP-binding protein aarF 4 N Probable ubiquinone biosynthesis protein accA 4 E Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha accB 4 E Biotin carboxyl carrier protein of acetyl-CoA carboxylase accC 4 E Biotin carboxylase aceE 4 N Pyruvate dehydrogenase E1 component acpP 4 E Acyl carrier protein ahpC 4 N Alkyl hydroperoxide reductase subunit C alaS 4 E Alanyl-tRNA synthetase apbE 4 U Thiamine biosynthesis lipoprotein apt 4 N Adenine phosphoribosyltransferase 152 atpA 4 N ATP synthase alpha chain atpD 4 N ATP synthase beta chain atpH 4 N ATP synthase delta chain bioD 4 N Dethiobiotin synthetase birA 4 N Biotin-[acetyl-CoA carboxylase] holoenzyme synthetase and repressor cca 4 N tRNA nucleotidyltransferase clpX 4 N ATP-dependent Clp protease ATP-binding subunit cutE 4 E Apolipoprotein N-acyltransferase cysB 4 N HTH-type transcriptional regulator dapA 4 N Dihydrodipicolinate synthase dapE 4 N Succinyl-diaminopimelate desuccinylase deaD 4 N Cold-shock DEAD-box protein A dgkA 4 N Diacylglycerol kinase dnaB 4 E Replicative DNA helicase dnaG 4 E DNA primase dnaJ 4 N Chaperone dnaK 4 N Chaperone dnaQ 4 N DNA polymerase III, epsilon chain dnaX 4 E DNA polymerase III subunit tau fabA 4 E 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase fabG 4 E 3-oxoacyl-[acyl-carrier-protein] reductase fabZ 4 E (3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase 153 ffh 4 E Signal recognition particle protein fis 4 N DNA-binding protein folA 4 N Dihydrofolate reductase ftsA 4 E Cell division protein ftsE 4 E Cell division ATP-binding protein ftsW 4 E Cell division protein ftsZ* 4 E Cell division protein fumC 4 N Fumarate hydratase class II fusA 4 E Elongation factor G galE 4 N UDP-glucose 4-epimerase galU 4 N UTP--glucose-1-phosphate uridylyltransferase gcpE 4 E 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase gcvA 4 N Glycine cleavage system transcriptional activator gidA 4 N Glucose inhibited division protein A glmM 4 E Phosphoglucosamine mutase glmU 4 E Bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-P uridyltransferase glnA 4 N Glutamine synthetase gloA 4 N Lactoylglutathione lyase gloB 4 U Probable hydroxyacylglutathione hydrolase greA 4 N Transcription elongation factor groL 4 E 60 kDa chaperonin grpE 4 E Hsp 24 DnaK nucleotide exchange factor 154 gshB 4 N Glutathione synthetase guaB 4 N Inosine-5'-monophosphate dehydrogenase hemC 4 U Porphobilinogen deaminase hemK 4 N Protein methyltransferase hemK hflC 4 N HflA complex cleaves lambda cII hflX 4 N GTP-binding protein hisD 4 N Histidinol dehydrogenase hisS 4 E Histidyl-tRNA synthetase htpX 4 N Probable protease ispA 4 E Geranyltranstransferase ispB 4 N Octaprenyl-diphosphate synthase kdsA 4 E 2-dehydro-3-deoxyphosphooctonate aldolase kdtB 4 E Phosphopantetheine adenylyltransferase lexA 4 N LexA repressor lgt 4 E Prolipoprotein diacylglyceryl transferase lolA 4 E Outer-membrane lipoprotein carrier protein lpd 4 N Dihydrolipoyl dehydrogenase lpxB 4 E Lipid-A-disaccharide synthase lpxD 4 E UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase lspA 4 E Lipoprotein signal peptidase lytB 4 E 4-hydroxy-3-methylbut-2-enyl diphosphate reductase menG 4 U Regulator of ribonuclease activity A 155 metB 4 N Cystathionine gamma-synthase metG 4 N Methionyl-tRNA synthetase mfd 4 N Transcription-repair coupling factor minD 4 N Septum site-determining protein mraY 4 E Phospho-N-acetylmuramoyl-pentapeptide-transferase mrcA 4 N Penicillin-binding protein 1A mrcB 4 N Penicillin-binding protein 1B mrdB 4 E Rod shape-determining protein mreB 4 N Rod shape-determining protein mreD 4 N Rod shape-determining protein murA 4 E UDP-N-acetylglucosamine 1-carboxyvinyltransferase murC 4 E UDP-N-acetylmuramate--L-alanine ligase murG 4 E UDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase murI 4 E Glutamate racemase mutS 4 N DNA mismatch repair protein mviN 4 U Virulence factor mviN homolog nth 4 N Endonuclease III nusB 4 E N utilization substance protein B nusG 4 E Transcription antitermination protein nusG pabA 4 N Para-aminobenzoate synthase glutamine amidotransferase component II pal 4 N Peptidoglycan-associated lipoprotein panC 4 N Pantoate--beta-alanine ligase 156 pdxA 4 N 4-hydroxythreonine-4-phosphate dehydrogenase pdxH 4 N Pyridoxamine 5'-phosphate oxidase pheS 4 E Phenylalanyl-tRNA synthetase alpha chain pheT 4 E Phenylalanyl-tRNA synthetase beta chain plsB 4 E Glycerol-3-phosphate acyltransferase pnp 4 N Polyribonucleotide nucleotidyltransferase priA 4 N Primosomal protein N' prs 4 E Ribose-phosphate pyrophosphokinase purB 4 N Adenylosuccinate lyase purH 4 N Bifunctional purine biosynthesis protein pyrF 4 N Orotidine 5'-phosphate decarboxylase pyrG 4 U CTP synthase rbfA 4 N Ribosome-binding factor A recC 4 N Exodeoxyribonuclease V gamma chain recN 4 N DNA repair protein recO 4 N DNA repair protein recQ 4 N ATP-dependent DNA helicase recR 4 N Recombination protein ribE 4 N 6,7-dimethyl-8-ribityllumazine synthase ribF 4 N Riboflavin biosynthesis protein rnd 4 N Ribonuclease D rnt 4 N Ribonuclease T 157 rplA 4 E 50S ribosomal protein L1 rplE 4 E 50S ribosomal protein L5 rplK 4 E 50S ribosomal protein L11 rplL 4 E 50S ribosomal protein L7/L12 rplO 4 E 50S ribosomal protein L15 rplQ 4 E 50S ribosomal protein L17 rplV 4 E 50S ribosomal protein L22 rplX 4 E 50S ribosomal protein L24 rpmC 4 E 50S ribosomal protein L29 rpmD 4 E 50S ribosomal protein L30 rpmE 4 E 50S ribosomal protein L31 rpmJ 4 E 50S ribosomal protein L36 rpoA 4 E DNA-directed RNA polymerase alpha chain rpoC 4 E DNA-directed RNA polymerase beta' chain rpoD* 4 E RNA polymerase sigma factor rpsD 4 E 30S ribosomal protein S4 rpsG 4 E 30S ribosomal protein S7 rpsH 4 E 30S ribosomal protein S8 ruvA 4 N Holliday junction DNA helicase sbcB 4 N Exodeoxyribonuclease I secA 4 E Preprotein translocase secA subunit secF 4 N Protein-export membrane protein 158 secY 4 E Preprotein translocase secY subunit slyX 4 N Protein slyX sohB 4 N Possible protease ssb 4 E Single-strand binding protein sucC 4 N Succinyl-CoA synthetase beta chain surE 4 N Acid phosphatase tatC 4 U Sec-independent protein translocase protein tatC thiL 4 U Thiamine-monophosphate kinase thrS 4 E Threonyl-tRNA synthetase thyA 4 N Thymidylate synthase tmk 4 E Thymidylate kinase tolQ 4 N TolQ protein tolR 4 N TolR protein trmU 4 E tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase trpB 4 N Tryptophan synthase beta chain truA 4 N tRNA pseudouridine synthase A tsf 4 U Elongation factor Ts typA 4 U GTP-binding protein tyrS 4 E Tyrosyl-tRNA synthetase ubiA 4 N 4-hydroxybenzoate octaprenyltransferase ubiG 4 N 3-demethylubiquinone-9 3-methyltransferase ubiH 4 N 2-octaprenyl-6-methoxyphenol hydroxylase 159 uvrA 4 N UvrABC system protein A uvrD 4 N DNA helicase II visC 4 N FAD-dependent oxidoreductase xseA 4 N Exodeoxyribonuclease VII large subunit xseB 4 N Exodeoxyribonuclease VII small subunit yabC 4 U S-adenosyl-methyltransferase yacE 4 E Dephospho-CoA kinase yadF 4 E Carbonic anhydrase 2 yadH 4 N Hypothetical transport permease yadR 4 U Hypothetical protein yaeS 4 E Undecaprenyl pyrophosphate synthetase yaeT 4 U Unknown protein from 2D-page spots M62/M63/O3/O9/T35 yaiD 4 U Recombination associated protein yajC 4 N Hypothetical protein ybaV 4 U Hypothetical protein ybbF 4 E UDP-2,3-diacylglucosamine hydrolase ybeA 4 U Hypothetical protein ybeX 4 U Magnesium and cobalt efflux protein ybgC 4 N Hypothetical protein ycbY 4 U Hypothetical protein yceG 4 U Hypothetical protein ycfC 4 E Hypothetical protein 160 ycfH 4 U Putative deoxyribonuclease ycfO 4 U Beta-hexosaminidase ycfV 4 E Lipoprotein releasing system ATP-binding protein ycfW 4 E Lipoprotein releasing system transmembrane protein ychB 4 E 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase ychF 4 N GTP-dependent nucleic acid-binding protein yciA 4 N Putative acyl-CoA thioester hydrolase yciO 4 N Hypothetical protein yebC 4 U Hypothetical protein yfcB 4 U Hypothetical adenine-specific methylase yfgM 4 U Hypothetical protein yfgO 4 N Putative permease yfhC 4 U Hypothetical protein yfhO 4 U Cysteine desulfurase yfiO 4 U Hypothetical lipoprotein ygbB 4 E 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase ygbP 4 E 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase ygbQ 4 E Cell division protein ygdL 4 N Hypothetical protein ygdP 4 U (Di)nucleoside polyphosphate hydrolase ygfY 4 U Hypothetical protein yhbG 4 U Probable ABC transporter ATP-binding protein 161 yhbJ 4 U Hypothetical protein yheS 4 U Hypothetical ABC transporter ATP-binding protein yhgF 4 N Hypothetical protein yhhF 4 U Putative methylase yhiN 4 U Hypothetical protein yicC 4 U Hypothetical protein yihZ 4 U D-tyrosyl-tRNA(Tyr) deacylase yjeQ 4 E Probable GTPase yjfH 4 U 23S rRNA (guanosine-2'-O-)-methyltransferase yjgA 4 N Hypothetical protein yjgF 4 U Hypothetical protein yjgP 4 U Hypothetical protein yjgQ 4 U Hypothetical protein yjjK 4 N ABC transporter ATP-binding protein yqgF 4 E Putative Holliday junction resolvase yraN 4 U Hypothetical protein yrbA 4 U Hypothetical protein yrbH 4 U Arabinose 5-phosphate isomerase yrbI 4 E 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase yrfH 4 U Heat shock protein 15 # *: Essentiality based on PEC, E:essential; N :non-essential; U: unknown. :The localization results of these gene products are previous published by our lab (29). 1:No clones on selective plates. 2: Located successfully. 3: Labelling successfully but no fluorescence signal. 4: Can not be tagged or located suceessfully by other reasons, like targetting cassette producing PCR failure, etc. 162 Appendix 3 Primers used in the study of protein localization of E. coli Name of oligo length of oligo (bases) Seuqnece of oligo (5' to 3') aarF-EGFP-Sense 87 TTAATGGCAGGTGGTCTGATCGCCTGGTTTGTCGGTTGGCGCAAAACACGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG aarF-EGFP-AntiSense 72 ACAAAGCCGCATTATACGTTACACGGCCCGCCTTGAGCGATGAAAAAATCATAGTGAACCTCTTCGAGGGAC aarF Sense Primer 20 TATTTTCTCGGAATTGGCGC aarF Antisense Primer 26 AATAACTGCCAAATACTGATACCACC abC-EGFP-Sense 87 ATTGCCTGGCTGCAGGAACACCATGTAAAAGTAGAGGTACTGGGTTATGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG abC-EGFP-AntiSense 72 ATTGCCAGCGTTTCCCATACGCCACGAACCAGCAGCCACATCATCGGCTCATAGTGAACCTCTTCGAGGGAC abC Sense Primer 20 CGGCATCATGCTGACTGAAA abC Antisense Primer 22 ACAAAGCCAAAAAAACCGGATA accA-EGFP-Sense 87 GAAGATTTAAAAAATCGTCGTTATCAGCGCCTGATGAGCTACGGTTACGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG accA-EGFP-AntiSense 72 CGATAAAAAAGGGCCACCGAAGTGACCCTTTTTCAGAACTTTTGCGAATTATAGTGAACCTCTTCGAGGGAC accA Sense Primer 19 TCGTTGAAAGCGCAACTGC accA Antisense Primer 24 TCCTTCCTTAATCATAGCCTGCTC accB-EGFP-Sense 87 GTCGAAAGTGGACAACCGGTAGAATTTGACGAGCCGCTGGTCGTCATCGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG accB-EGFP-AntiSense 72 ATCTCGCCGCGGTTGGCAATAACAATTTTATCCAGCATGTTCGCCTCGTTATAGTGAACCTCTTCGAGGGAC accB Sense Primer 20 CCAGATCGAAGCGGACAAAT accB Antisense Primer 24 TTCTTTACAGGCACGAAGAATACG accC-EGFP-Sense 87 GGTGGCACTAACATCCACTATCTGGAGAAAAAACTCGGTCTTCAGGAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG accC-EGFP-AntiSense 72 AGTAATAAAAAAGGCCGGAAAATCCGGCCTTTTGACGCTTTAGCAGTCTTATAGTGAACCTCTTCGAGGGAC accC Sense Primer 20 CCGCATCATGAATGACGAGA accC Antisense Primer 21 AGCGGGGATTGTACCTTATGG accD-EGFP-Sense 87 CCGCGTGAAGGCGTAGTGGTACCCCCGGTACCGGATCAGGAACCTGAGGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG accD-EGFP-AntiSense 72 GTGAGAATAGCAAAAGGGCAGAGCCAGTGGCCCTGCCCTTATCAGTTATCATAGTGAACCTCTTCGAGGGAC accD Sense Primer 19 CCAGCGCAGTGAATTCCTG accD Antisense Primer 24 TGATAATCATGGTATCCGCTGATT aceE-EGFP-Sense 87 GCAATCGCCAAATTCAACATCGATGCAGATAAAGTTAACCCGCGTCTGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 163 aceE-EGFP-AntiSense 72 CCCCGATGTCCGGTACTTTGATTTCGATAGCCATTATTCTTTTACCTCTTATAGTGAACCTCTTCGAGGGAC aceE Sense Primer 24 AATCGATAAGAAAGTGGTTGCTGA aceE Antisense Primer 23 GGATCTCGGTGATTTCAACTTCA aceF-EGFP-Sense 87 TTCATTACCATCATTAACAACACGCTGTCTGACATTCGCCGTCTGGTGATGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG aceF-EGFP-AntiSense 72 TTCATGAGATTACCAGAAAAAAGCCGGCCGTTGGGCCGGCTCTTTTACTTATAGTGAACCTCTTCGAGGGAC aceF Sense Primer 19 TGATCGACGGTGCTGATGG aceFAntisense Primer 22 AACGGCAACCGATTTGTCTATT acpP-EGFP-Sense 87 AAAATCACCACCGTTCAGGCTGCCATTGATTACATCAACGGCCACCAGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG acpP-EGFP-AntiSense 72 CAAAAAGATAAAACTCAGGCGGTCGAACGACCGCCTGGAGATGTTCACTTATAGTGAACCTCTTCGAGGGAC acpP Sense Primer 23 GATACTGAGATTCCGGACGAAGA acpP Antisense Primer 24 CAGGGAGGGAAAAAATGATTCTAG ahpC-EGFP-Sense 87 AAAGAAGGTGAAGCAACTCTGGCTCCGTCTCTGGACCTGGTTGGTAAAATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ahpC EGFP-Antisense 72 GTGAGCGGGCGACGCCAACGCCGCTATGGCGTGAAAGACGAAGGAAATTTATAGTGAACCTCTTCGAGGGAC ahpC Sense Primer 19 GCGTCTGACCTGCTGCGTA ahpC Antisense Primer 23 GAAGTATTTCGCGTCAGAGCAAG alaS-EGFP-Sense 87 TTACCTGCAGCGTTAGCCAGTGTGAAAGGCTGGGTCAGCGCGAAATTGCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG alaS-EGFP-AntiSense 72 TAATGCGAATGCCGTGAAGCGAGTCCACGGCATTGCCTGACGCTTATATTATAGTGAACCTCTTCGAGGGAC alaS Sense Primer 20 GGTGGTGGACGTCCTGACAT alaS Antisense Primer 24 CTGACTTTATCGTTGTCGATAGCG apaH-EGFP-Sense 87 TTTGTCCAGCCGTCGAACCGGCATAAGGATTTGGGCGAAGCGGCGGCGTCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG apaH-EGFP-AntiSense 72 TGCGACGCCGGTCGCGTCTTATCCGGCCTTCCTATATCAGGCTGTGTTTATAGTGAACCTCTTCGAGGGAC apaH Sense Primer 21 CTGCGCTGGGAAGATAAACAG apaH Antisense Primer 21 TAGAATTTACGGCTAGCGCCG apbE-EGFP-Sense 87 AAAACCTGGATGTCACCACAGTTTAAAAGCTTCCTTGTCAGCGAAAAAAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG apbE-EGFP-AntiSense 72 CTTTAGGCTGCCCGGTCACCATCACGCAAAAACCAACAATCTTGCGCTTTATAGTGAACCTCTTCGAGGGAC apbE Sense Primer 24 GCGGTTTATATGATCACCAAAGAA apbE Antisense Primer 23 CATAATCAGCTCCCTGGTTAAGG apT-EGFP-Sense 87 CTCGAAAAACAGGGCATTACCAGCTACAGCCTTGTCCCGTTCCCGGGCCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 164 apT-EGFP-AntiSense 72 GCACATCTTGTGCTGTCCGTAGCGTGGGCAGCACAAGACTGGCGATAATTATAGTGAACCTCTTCGAGGGAC apT Sense Primer 23 GCTGCGTTCATTATCAACCTGTT apT Antisense Primer 21 GGATTCACGAAGGGGTAATGC aroB-EGFP-Sense 87 GGCGTTTCGCACGAGCTTGTTCTTAACGCCATTGCCGATTGTCAATCAGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG aroB-EGFP-AntiSense 72 ACCTGAAGCTAATAGACCGCTTGATAAGCGGCCTGACCTTTCTTGTTGTTATAGTGAACCTCTTCGAGGGAC aroB Sense Primer 20 ATTCTTCCGTTGGCAATTGG aroB Antisense Primer 23 AAGGTTAATGCTTACCACGTTGC aroC-EGFP-Sense 87 TTACGGCAACGGGCGCAAAATGCCGATGTGAAGACTGATATTCCACGCTGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG aroC-EGFP-AntiSense 72 GCTACTGGCAAGCAGAGCCAGCAGCGCAATCGCGGTTTTATTCATTTTTTATAGTGAACCTCTTCGAGGGAC aroC Sense Primer 19 GATCGCAGAAGCGATGCTG aroC Antisense Primer 20 CGGCACAGGTTGGGTTATTT aroE-EGFP-Sense 87 CTGCCTGACGTAGAACCAGTTATAAAGCAATTGCAGGAGGAATTGTCCGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG aroE-EGFP-AntiSense 72 TTTTATTCTCGTCCCACTCTTCCCTGTCCGGAAACTGGATGGCCTGATTCATAGTGAACCTCTTCGAGGGAC aroE Sense Primer 22 CTTTCTTCTCTGGCACGGTGTT aroE Antisense Primer 21 TCACGAGAGCGGGAAAACATA aroK-EGFP-Sense 87 CAAAGCGCTAAAGTGGTTGCAAACCAGATTATTCACATGCTGGAAAGCAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG aroK-EGFP-AntiSense 72 ATCCACCTTAATTACTGTACCCGCAGACGAGTGTATATAAAGCCAGAATTATAGTGAACCTCTTCGAGGGAC aroK Sense Primer 19 GAGATTGCCGACGTGACCA aroK Antisense Primer 22 ACGACAATCCTCTCCATAACGC aspS-EGFP-Sense 87 ACTGCACTGGCTGAGCTGAGCATTCAGGTTGTGAAGAAGGCTGAGAATAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG aspS-EGFP-AntiSense 72 AGTTGCCGCCTCGATGCAACGCGAATGATTTCGTGTATTTGAGTCATATCATAGTGAACCTCTTCGAGGGAC aspS Sense Primer 20 ACTGAAGCACCGAGCTTTGC aspS Antisense Primer 20 CCTTCGCTCATCCCATTCAC atpA-EGFP-Sense 87 GAAGGCAAGCTGAAAGGCATCCTCGATTCCTTCAAAGCAACCCAATCCTGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG atpA-EGFP-AntiSense 72 GCTTCTCCTCAATGCCTTGCGGCCTGCCCTAAGGCAAGCCGCCAGACGTTATAGTGAACCTCTTCGAGGGAC atpA Sense Primer 20 AGACCGGTGGCTACAACGAC atpA Antisense Primer 21 CTACGTATCTCTTTTGCGCCG atpB-EGFP-Sense 87 ATCTTCATGGTTCTGACGATCGTCTATCTGTCGATGGCGTCTGAAGAACATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 165 atpB-EGFP-AntiSense 72 ATGACAGTCTCCAGTTTGTTTCAGTTAAAACGTAGTAGTGTTGGTAAATTATAGTGAACCTCTTCGAGGGAC atpB Sense Primer 22 TTCCACATCCTGATCATTACGC atpB Antisense Primer 26 ATGTACAGCAGATCCATATTCAGGTT atpC-EGFP-Sense 87 GCCAAAGCGATCGCGCAGCTGCGCGTTATCGAGTTGACCAAAAAAGCGATGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG atpC-EGFP-AntiSense 72 AAAAGCCAGCCTGTTTCCAGACTGGCTTTTGTGCTTTTCAAGCCGGTGTTATAGTGAACCTCTTCGAGGGAC atpC Sense Primer 21 CGTAGATTACGCTCAGGCGTC atpC Antisense Primer 20 CGCTATTCAGGACGGGTCAC atpD-EGFP-Sense 87 TTCTACATGGTCGGTTCCATCGAAGAAGCTGTGGAAAAAGCCAAAAAACTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG atpD-EGFP-AntiSense 72 CGACGTCCAGGTGGTAAGTCATTGCCATATCACCCTCCGATTAAGGCGTTATAGTGAACCTCTTCGAGGGAC atpD Sense Primer 18 GGCATCATGGAAGGCGAA atpD Antisense Primer 21 GAACATTTGTTGCTCTGCGCT atpE-EGFP-Sense 87 ATCCCGATGATCGCTGTAGGTCTGGGTCTGTACGTGATGTTCGCTGTCGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG atpE-EGFP-AntiSense 72 TATTTAGTTAACGTTCTGATATTGCTCTTTAAATAAAAGCAACGCTTACTATAGTGAACCTCTTCGAGGGAC atpE Sense Primer 24 TTCTTTATCGTTATGGGTCTGGTG atpE Antisense Primer 23 GTTGCGTTAAGATTCACAGCACA atpF-EGFP-Sense 87 GTGGATGAAGCTGCTAACAGCGACATCGTGGATAAACTTGTCGCTGAACTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG atpF-EGFP-AntiSense 72 CGTAGGGGCGAGCTACCGTAATAAATTCAGACATCAGCCCCTCCCTCCTTATAGTGAACCTCTTCGAGGGAC atpF Sense Primer 22 CAAGTTGCTATCCTGGCTGTTG atpF Antisense Primer 20 ACGGCAAAGTCAAAAGCTGC atpG-EGFP-Sense 87 GCCAGCATTACTCAGGAACTCACCGAGATCGTCTCGGGGGCCGCCGCGGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG atpG-EGFP-AntiSense 72 GGACAATCTTTCCAGTAGCCATCTTAAATCCTCTACGAAATAACCTGTTTATAGTGAACCTCTTCGAGGGAC atpG Sense Primer 24 GCAGTTGGTATACAACAAAGCTCG atpG Antisense Primer 19 TTCGACGTCAACTACGGCG atpH-EGFP-Sense 87 GATGGCAGCGTACGCGGTCGTCTTGAGCGCCTTGCAGACGTCTTGCAGTCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG atpH-EGFP-AntiSense 72 GATCAGTTCGCTGATTTCGGTGGAATTCAGTTGCATGCTCCAGTCCCCTTATAGTGAACCTCTTCGAGGGAC atpH Sense Primer 23 ATAAGTCTGTAATGGCAGGCGTT atpH Antisense Primer 23 CACAACATTGAACTGAGCAATGC bcP-EGFP-Sense 87 AAAACCAGCAATCACCACGACGTTGTGCTGAACTGGCTGAAAGAACACGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 166 bcP-EGFP-AntiSense 72 GAGGTATGCTGGCCGCAAGCGCAGCCAGCACGGAATGGAGCAAAGTAATCATAGTGAACCTCTTCGAGGGAC bcP Sense Primer 20 CTGATTGACGCTGATGGCAA bcP Antisense Primer 20 AAATGGATTCGACACCCGTG bcR-EGFP-Sense 87 GCAACCAGCTCCATTCTCTTCTGTCTGTACGCCAGTCGGCCGAAAAAACGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG bcR-EGFP-AntiSense 72 ACATGCATCAACAAAGGAAGCCTTTTAGCTTCCTCGTTGTGCAATAGATCATAGTGAACCTCTTCGAGGGAC bcR Sense Primer 22 CGATGATTTGGTCAATTGCATT bcR Antisense Primer 23 CCGTTTCTGCGATCTAACTCAAC bioA-EGFP-Sense 87 CTGACCGCAGCGGTTAACCGCGCGGTACAGGATGAAACATTTTTTTGCCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG bioA-EGFP-AntiSense 72 TTCTTGTTTGCAGAAAGTGTAGCCAGAAACCCTCACGCGGACTTCTCGTTATAGTGAACCTCTTCGAGGGAC bioA Sense Primer 21 CGCCCTATATTATTCTCCCGC bioA Antisense Primer 23 CTGATGAGTTTCATGAACCCTCC bioB-EGFP-Sense 87 CAGGCGCTGATGACCCCGGACACCGACGAATATTACAACGCGGCAGCATTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG bioB-EGFP-AntiSense 72 CGGCAGCACGCCGCGCATCGAGCGCCGCGTTGATTTTCTCCTGCCAGCTCATAGTGAACCTCTTCGAGGGAC bioB Sense Primer 22 GGGGATAACGAACAACAGCAAC bioB Antisense Primer 21 TCAGATACTGGCGATCATCCG bioD-EGFP-Sense 87 GAAAATCCAGAAAATGCGGCAACCGGAAAGTACATAAACCTTGCCTTGTTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG bioD-EGFP-AntiSense 72 TCAATCGAAGACGCGATCTCGCTCGCAATTTAACCAAATACAGAATGGCTATAGTGAACCTCTTCGAGGGAC bioD Sense Primer 22 CGCTGAATATATGACCACGCTC bioD Antisense Primer 18 CCGCATGAGGCGGAAGTA bioF-EGFP-Sense 87 CATGAAATGCAGGATATCGACCGTCTGCTGGAGGTGCTGCATGGCAACGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG bioF-EGFP-AntiSense 72 TCATAGTGTGCGGCTGCCCGACCAAATGCCGCTGCAATGGCTTGTTTATTATAGTGAACCTCTTCGAGGGAC bioF Sense Primer 20 ACTGCGCTTAACGCTAACCG bioF Antisense Primer 21 TCTGGCGCTGTAGATCTGCAT birA-EGFP-Sense 87 ATAATAAAACCCTGGATGGGCGGTGAAATATCCCTGCGTAGTGCAGAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG birA-EGFP-AntiSense 72 AGGTCAGACTACGCAAATAATTTGCAGGGGAGCGAATACTCCCCTTTCTTATAGTGAACCTCTTCGAGGGAC birA Sense Primer 20 CGCGGAATAGACAAACAGGG birA Antisense Primer 21 CGAAAAGTGCTAATCATGCGG bolA-EGFP-Sense 87 CAGGACACCGTCTTTGCCTCTCCTCCCTGTCGTGGAGCAGGAAGCATCGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 167 bolA-EGFP-AntiSense 72 AATCTTTAGCAACATACTGGAAAAGCGCCGACAGTTGCAAATGCGTTTTTATAGTGAACCTCTTCGAGGGAC bolA Sense Primer 25 ACACTATTAAGGAGTGGGAAGGGTT bolA Antisense Primer 21 AAAAAGCGCAAAATCCAGACA cafA-EGFP-Sense 87 GTACAAATTGAACCGCTCTATAACCAGGAGCAGTTTGACGTCGTAATGATGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cafA-EGFP-AntiSense 72 AAAAATGTGACTCAAAAACCCTTTGCCGGATGGCGGCCCAGCATCTGTTTATAGTGAACCTCTTCGAGGGAC cafA Sense Primer 21 TGGAAATTTTCGTTGGCAAAC cafA Antisense Primer 21 TCACCCGTCACTCCTTGTCTG carA-EGFP-Sense 87 TTGTTCGACCACTTTATCGAGTTAATTGAGCAGTACCGTAAAACCGCTAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG carA-EGFP-AntiSense 72 GGATACTTTTTATATCTGTACGTTTTGGCATGGCTCTTTTACTCCTGATTATAGTGAACCTCTTCGAGGGAC9 carA Sense Primer 21 ATAAACCGGCATTCAGCTTCC carA Antisense Primer 20 CGCCTGACCGATAACAATCG carB-EGFP-Sense 87 GATGCGACTGAAAAAGTAATTTCGGTGCAGGAAATGCACGCACAGATCAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG carB-EGFP-AntiSense 72 GTTATTCGATCAAATTAGCGGATGAAAAATATCTGCCATGACACGCTATTATAGTGAACCTCTTCGAGGGAC carB Sense Primer 20 CGCAGTGCGCTGCAATATAA carB Antisense Primer 21 ACGGTCCTCATCAGAGAACCG ccA-EGFP-Sense 87 CGGATTGCGGCGGTAGCCAGCTGGAAGGAACAACGTTGCCCAAAGCCTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ccA-EGFP-AntiSense 72 TGTAGCCGGATAAGGCGTTTACGCCGCATCCGGCAAAATGCCCAATACTCATAGTGAACCTCTTCGAGGGAC ccA Sense Primer 19 GTGGAGATTCGCGAGGAGC ccA Antisense Primer 19 CGCTTGCGCGTCTTATCAG ccmA-EGFP-Sense 87 GTTGCTGAAAGTAAAATTCGCCGCATTTCACTGACGCAAACGAGGGCCGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ccmA-EGFP-AntiSense 72 TATGGCGAAACGCTACACGCAGCTCAAGACGGAAAATGCGCCAGAACATCATAGTGAACCTCTTCGAGGGAC ccmA Sense Primer 22 TGATTCTGACTACCCACCAGCC ccmA Antisense Primer 22 ACAATCAGGAAGAACCACAGCG ccmB-EGFP-Sense 87 GCGACATTAAGTCCTTTTGCGACGGCGGCAGCGTTACGAATCAGCATTCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ccmB-EGFP-AntiSense 72 TCCACATAGTTTCGATACCAGACTCGAACAAAAATCAGTAATCCAGCGTTATAGTGAACCTCTTCGAGGGAC ccmB Sense Primer 22 GTTGACGGGTATCTGGCAATTT ccmB Antisense Primer 20 GGATCGCCAGTTGATGCAGT ccmC-EGFP-Sense 87 GAAAAACGCCGTCCGTGGGTGAGTGAACTGATACTGAAAAGAGGCCGTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 168 ccmC-EGFP-AntiSense 72 CGTAACCGCCCATTGCGAAAAATTCATTCCAGGAAGCAAATGCAGGGGTCATAGTGAACCTCTTCGAGGGAC ccmC Sense Primer 21 TTTGGCTTCCTGCTCCTGTCT ccmC Antisense Primer 19 ACCAAAACCACCAGCGGAA ccmE-EGFP-Sense 87 ATGGAAGCTAACCACCGTCGCCCGGCGAGTGTTTATAAGGACCCAGCATCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ccmE-EGFP-AntiSense 72 GCGCAATTCCCAGCGCCAGGCACAGCAGTCCGTTACCAATTTCTGGCATCATAGTGAACCTCTTCGAGGGAC ccmE Sense Primer 19 GCTGGCGAAACACGATGAA ccmE Antisense Primer 21 ATAGCGGATACACGGACAGCA ccmF-EGFP-Sense 87 CCTCGCTATCGTAAGCGCGTGAGTCCGCAAAAAACTGCGCCGGAGGCCGTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ccmF-EGFP-AntiSense 72 CGGCAATCGCCAGGAAGATAATCAACGGAATTAACAATACTTTGCGCTTCATAGTGAACCTCTTCGAGGGAC ccmF Sense Primer 22 TACAAACCATTTGTTCGCTGGA ccmF Antisense Primer 22 GATTCCAGATTGGTCGGATCAT ccmG-EGFP-Sense 87 GAAGAAGAGATCAAGCCGCTGTGGGAGAAATACAGTAAGGAGGCCGCACAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ccmG-EGFP-AntiSense 72 CCAGCGCTGAGCCGGAGATCATCAGCATCAGCACGCCCAATAAAAACCTCATAGTGAACCTCTTCGAGGGAC ccmG Sense Primer 20 GCAACGGCATCATTCGCTAT ccmG Antisense Primer 22 AGCTGACGGAACTGTTGTTCCT cdsA-EGFP-Sense 87 GCTGCGGTACCGGTCTTTGCTTGCTTGTTGTTACTGGTATTCAGGACGCTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cdsA-EGFP-AntiSense 72 CGATGAACGAAGCCAAATCCCAGAGAAAACTCAGCATATTACCTTCCGTTATAGTGAACCTCTTCGAGGGAC cdsA Sense Primer 21 GCAGGAATTAAGGACAGCGGT cdsA Antisense Primer 21 ACCAAATTCATGCACGGTGAT clpB-EGFP-Sense 87 GGTAAAGTGATTCGCCTGGAAGTTAATGAAGACCGGATTGTCGCCGTCCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG clpB-EGFP-AntiSense 72 CGTCTAACTTATAGACAAAAACGAGCCCCGAAGGGCTCGTTTTATCATTTATAGTGAACCTCTTCGAGGGAC clpB Sense Primer 22 TGGCACAGCAAATACTGTCTGG clpB Antisense Primer 24 GTGGCGCATTATAGGGAGTTATTC clpP-EGFP-Sense 87 CCTGAAGCGGTGGAATACGGTCTGGTCGATTCGATTCTGACCCATCGTAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG clpP-EGFP-AntiSense 72 CGTTGTGCCGCCCTGGATAAGTATAGCGGCACAGTTGCGCCTCTGGCATCATAGTGAACCTCTTCGAGGGAC clpP Sense Primer 20 TTATGGCGCTTCATACGGGT clpP Antisense Primer 21 CTTCTTTGTTCTTTGTGCCGC clpX-EGFP-Sense 87 AAACCGTTGCTGATTTATGGCAAGCCGGAAGCGCAACAGGCATCTGGTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 169 clpX-EGFP-AntiSense 72 AGATAAAATCCCCCCTTTTTGGTTAACTAATTGTATGGGAATGGTTAATTATAGTGAACCTCTTCGAGGGAC clpX Sense Primer 21 ATGTACGATCTGCCGTCCATG clpX Antisense Primer 22 CAACGCCGAGAATAGAGGAAAA cmK-EGFP-Sense 87 CAAGTGATTGAAAAAGCGCTACAATACGCGCGCCAGAAATTGGCTCTCGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cmK-EGFP-AntiSense 72 ATACCCGCTGTCATTCCATTGCAACGGGGGTACTGCAAATTCGGTCGCTTATAGTGAACCTCTTCGAGGGAC cmK Sense Primer 23 AGTGTTGGATTCCACCACCTTAA cmK Antisense Primer 19 AACGTCCACCTGGCTCCAT crcB-EGFP-Sense 87 TTTGCCATGACCGCACTGGCATTCTGGCTGTTTTCGGCCTCAACCGCACACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG crcB-EGFP-AntiSense 72 GTCAGCAAGAATTCAAAACCCGCTTAATCAGCGGGTTTTTTTTGGTCTTTATAGTGAACCTCTTCGAGGGAC crcB Sense Primer 23 CTGAACGTTTTCGTCAACCTTCT crcB Antisense Primer 20 TGCTGCAAACGTAATCGCTC csrA-EGFP-Sense 87 GAAGAGATCTACCAGCGTATCCAGGCTGAAAAATCCCAGCAGTCCAGTTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG csrA-EGFP-AntiSense 72 AGAAATTTTGAGGGTGCGTCTCACCGATAAAGATGAGACGCGGAAAGATTATAGTGAACCTCTTCGAGGGAC csrA Sense Primer 22 CCCGAAGGAAGTTTCTGTTCAC csrA Antisense Primer 22 TTGAATGAACGGGAGTAAAGCG cutE-EGFP-Sense 87 GCATTGTTTGGTTTTGCTGCTGTGTTGATGAGTCTGCGTCAGCGACGTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cutE-EGFP-AntiSense 72 GGTGAGCGTGCCGAAATATGGGATGTATTCCGGCACGATAAGAAGGGATTATAGTGAACCTCTTCGAGGGAC cutE Sense Primer 24 GAGGTGTTAACCACTAACGTGACG cutE Antisense Primer 22 TATCCGGATAATGCACTGATGC cydA-EGFP-Sense 87 ACCGGTCGCTATCACTTTGAGCAGTCTTCCACGACTACTCAGCCGGCACGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cydA-EGFP-AntiSense 72 CCAGATAAAACGCAATACTTCATAATCGATCATTTGACGACTCCTGTCTTATAGTGAACCTCTTCGAGGGAC cydA Sense Primer 22 TTAATGTTCAAGTTTGCACGCC cydA Antisense Primer 19 TCAGCAGAACGCCAACCAG cydB-EGFP-Sense 87 TTCGGTCGTATCACCAAAGAAGATATTGAACGTAACACCCACTCTCTGTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cydB-EGFP-AntiSense 72 GAAGCGTTCCCAGAATCCATGCGAAATACCACATTTTTAGCTCCTTACTTATAGTGAACCTCTTCGAGGGAC cydB Sense Primer 23 GTTCTGGTACCGATCATTCTGCT cydB Antisense Primer 20 CGGTGATTACCCCAAACGAA cysB-EGFP-Sense 87 TCTAATGAAGAAATTGAGGTCATGTTTAAAGATATAAAACTGCCGGAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 170 cysB-EGFP-AntiSense 72 ATTCCGGCACCTTCGCTACATAAAAGGTGCCGAAAATAACGCAAGAAATTATAGTGAACCTCTTCGAGGGAC cysB Sense Primer 20 GCATTTAACGCGTGATGTCG cysB Antisense Primer 24 CGAGGCGGGTAATTAGACACTACT cysK-EGFP-Sense 87 TTAAGCACCGCATTGTTTGCCGATCTCTTCACTGAGAAAGAATTGCAACAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cysK-EGFP-AntiSense 72 AAAAGCACCTAAAAAGGTGCTTTTTTACGCATTTTTAACAAGCTGGCATTATAGTGAACCTCTTCGAGGGAC cysK Sense Primer 24 GTGGTTATTCTACCATCATCGGGT cysK Antisense Primer 20 GGAGGGGCGAAAGTTTGAAG cysQ-EGFP-Sense 87 TACACTCCGCGTGAGTCGTTCCTGAATCCGGGGTTCAGAGTGTCTATTTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cysQ-EGFP-AntiSense 72 AAATCAGGATGGCAGAATCAGGAAATACACTGTTTCTGCCATCTGAATTTATAGTGAACCTCTTCGAGGGAC cysQ Sense Primer 21 TTCACGACTGGCAGGGTAAAC cysQ Antisense Primer 25 TTTGGTTACTTTATGTCGCCATTAA cysS-EGFP-Sense 87 ATGGGGATCGTGCTGGAAGATGGCCCGCAAGGGACCACCTGGCGTCGTAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG cysS-EGFP-AntiSense 72 GCGCAGACGATAACCGGATGCGAAAACTCGCATCCGGCAATAGCGCAATTATAGTGAACCTCTTCGAGGGAC cysS Sense Primer 21 AATTCAACAGCGTCTGGATGC cysS Antisense Primer 23 GGATGCTACTGATGGGAATGTTG dapA-EGFP-Sense 87 GACAGTGGTCGTGAGACGGTCAGAGCGGCGCTTAAGCATGCCGGTTTGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dapA-EGFP-AntiSense 72 GCCAGGCGCGACTTTTGAACAGAGTAAGCCATCAAATCTCCCTAAACTTTATAGTGAACCTCTTCGAGGGAC dapA Sense Primer 22 GGTGAAATGGGCATGTAAGGAA dapA Antisense Primer 22 AATAAAACAAGCGAAACACCCG dapB-EGFP-Sense 87 AAGGAAAGCGGTCTTTTTGATATGCGAGATGTACTTGATCTCAATAATTTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dapB-EGFP-AntiSense 72 TCAATAAATTAAATGTGTTATTTTTGCACCATAACAAATATTTTGTGGTTATAGTGAACCTCTTCGAGGGAC dapB Sense Primer 22 GGTAAGATCGGCTTTGTGGTTG dapB Antisense Primer 21 AGGGCCAAAAATTAAAGCCCT dapE-EGFP-Sense 87 CTGCAGCTACTTGCCCGTATGTATCAACGTATCATGGAACAGCTCGTCGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dapE-EGFP-AntiSense 72 ATATTTAGCCAGCCAGTCCATGCTTATTTCCTCTTGCAGAACCACTCATCATAGTGAACCTCTTCGAGGGAC dapE Sense Primer 20 GCCGGTCAATGCCACTATTC dapE Antisense Primer 24 CCTACCAAAAAGACAATCACCAGA dapF-EGFP-Sense 87 TTATATATGACTGGCCCGGCGGTACATGTCTACGACGGATTTATTCATCTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 171 dapF-EGFP-AntiSense 72 GGTCATCAAGCTCCGTGAGTGTTTCCTGCAGTTCTTCCCCTGGTTGCTTCATAGTGAACCTCTTCGAGGGAC dapF Sense Primer 22 TCTTGATATCGCCTGGAAAGGT dapF Antisense Primer 22 AATCAGATAATCGACAACCGCC deaD-EGFP-Sense 87 GCTCCGCGTCGTGATGATTCTACCGGTCGTCGTCGTTTCGGTGGTGATGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG deaD-EGFP-AntiSense 72 GGGGCTGTATATATTATTTTACAGATTGTGTTCGCTGTTCAGCGATGATTATAGTGAACCTCTTCGAGGGAC deaD Sense Primer 19 TCGTTTTAGCGGCGAACGT deaD Antisense Primer 22 AAAAAGCCCCGATGGTAAAAAT deF-EGFP-Sense 87 CAACGTATTCGTCAGAAAGTTGAAAAACTGGATCGTCTGAAAGCCCGGGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG deF-EGFP-AntiSense 72 TACCCGCAAAAATAATACGTAGTGATTCTGACACGTTAGTTCTTATCCTTATAGTGAACCTCTTCGAGGGAC deF Sense Primer 23 ATTCAGCATGAGATGGATCACCT deFAntisense Primer 21 GCCAACGACGTTATGACCAGA dfP-EGFP-Sense 87 CAATTATTACTCGACGAGATCGTGACCCGTTATGATGAAAAAAATCGACGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dfP-EGFP-AntiSense 72 GCATAAGTCGGGAGCGGAAATTCCTTCCCAACGCGCGGGTCCAGAATCTTATAGTGAACCTCTTCGAGGGAC dfP Sense Primer 20 CTTGAGCGCAAAGAGCTCCT dfP Antisense Primer 19 GAGACAGGCACGCAGGTCA dgkA-EGFP-Sense 87 ATTATCGTCGCCGTGATTACCTGGTGCATTCTGTTATGGTCGCATTTTGGACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dgkA-EGFP-AntiSense 72 AACCATAAACTGCACAATAAACCAGAGATTTATCGAATTCTGGAAGGGTTATAGTGAACCTCTTCGAGGGAC dgkA Sense Primer 21 AAAAGATATGGGATCCGCTGC dgkA Antisense Primer 25 CTGTGAGTATATACAGCAAAAGGCG dksA-EGFP-Sense 87 TGCATCGACTGCAAAACGCTGGCTGAAATTCGCGAAAAACAGATGGCTGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dksA-EGFP-AntiSense 72 AGGCGGGAGCATTTCCCGCCTGTGGTAAACGTGATGGAACGGCTGTAATTATAGTGAACCTCTTCGAGGGAC dksA Sense Primer 21 ATTGGTATTCGCCGTCTGGAA dksA Antisense Primer 20 GGCGAAGCGGCCAATATACT dnaA-EGFP-Sense 87 AGCCACGATATCAAAGAAGATTTTTCAAATTTAATCAGAACATTGTCATCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dnaA-EGFP-AntiSense 72 TGTAGCGGTTTTAATAAATGCTCACGTTCTACGGTAAATTTCATAGGTTTATAGTGAACCTCTTCGAGGGAC dnaA Sense Primer 21 AAGATCGAGCAGTTGCGTGAA dnaA Antisense Primer 22 TACCGAGAATCGGTAGCGTAGG dnaB-EGFP-Sense 87 GGTCAATGGTCGCGCTTCGACAACTATGCGGGGCCGCAGTACGACGACGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 172 dnaB-EGFP-AntiSense 72 GTTCCTTGATAAGTGTTTGCTTTAATTACCTAATTCATAAAATAATTATTATAGTGAACCTCTTCGAGGGAC dnaB Sense Primer 21 GTAAACAACGTAACGGCCCAA dnaB Antisense Primer 20 ACAACAGTTGCCGCTTGCAT dnaE-EGFP-Sense 87 AACGATCTCCGTGGCCTCATTGGTTCGGAGCAGGTGGAACTGGAGTTTGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dnaE-EGFP-AntiSense 72 AATCGGCTGTTCAAAATCAAGGAAATTCAGACTCATAGTATTCCTGTATTATAGTGAACCTCTTCGAGGGAC dnaE Sense Primer 21 TGTCTCTCCGAGCGATCGTTT dnaE Antisense Primer 20 CGGCTAACCGCAGTCAGAGA dnaG-EGFP-Sense 87 AACGAAGAACGCCTGGAGCTCTGGACATTAAACCAGGAGCTGGCGAAAAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dnaG-EGFP-AntiSense 72 CGGCTGTCGGGGGCTTCCCGATCGCTCTTCGGCACTTAAGCCGTTAAATCATAGTGAACCTCTTCGAGGGAC dnaG Sense Primer 21 CGCCAGGAAGAGTTAATCGCT dnaG Antisense Primer 22 ATATATTTGCCGCTGCCTCTCA dnaJ-EGFP-Sense 87 TCAAAGAGCTTCTTTGATGGTGTGAAGAAGTTTTTTGACGACCTGACCCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dnaJ-EGFP-AntiSense 72 GCACCCTATTTTTACCCAGGCCTGCCCACGGGCAGGCTTTTGGGGAGGTTATAGTGAACCTCTTCGAGGGAC dnaJ Sense Primer 21 TGCAAGAGCTGCAAGAAAGCT dnaJ Antisense Primer 25 ACTTTACAGGTGCTCGCATATCTTC dnaK-EGFP-Sense 87 AAAGATGACGATGTTGTCGACGCTGAATTTGAAGAAGTCAAAGACAAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dnaK-EGFP-AntiSense 72 GAAATTCCCCTTCGCCCGTGTCAGTATAATTACCCGTTTATAGGGCGATTATAGTGAACCTCTTCGAGGGAC dnaK Sense Primer 18 CTGCCGGTGCTGATGCTT dnaK Antisense Primer 20 TCACGCTCTTCCGCTGTTTT dnaN-EGFP-Sense 87 GAAGATGCGGCCAGCCAGAGCGCGGCTTATGTTGTCATGCCAATGAGACTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dnaN-EGFP-AntiSense 72 CGGTTTCAATGTTGCGGAAATCGCGGATCAACAAGCGGGTGAGGGACATTATAGTGAACCTCTTCGAGGGAC dnaN Sense Primer 20 AAAACGTCCGCATGATGCTG dnaN Antisense Primer 20 CCGTTGGCACCTACCAGAAA dnaQ-EGFP-Sense 87 GCCCGTCTCGATCTGGTGCAGAAGAAAGGCGGAAGTTGCCTCTGGCGAGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dnaQ-EGFP-AntiSense 72 GCTGCAAAAATCGCCCAAGTCGCTATTTTTAGCGCCTTTCACAGGTATTTATAGTGAACCTCTTCGAGGGAC dnaQ Sense Primer 24 ACAGATGAAGAGATTGCAGCTCAT dnaQ Antisense Primer 23 ATATTACGGATTGCCTCGACCTT dnaX-EGFP-Sense 87 CTGCGTCGGTTCTTCGATGCGGAGCTGGATGAAGAAAGTATCCGCCCCATTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 173 dnaX-EGFP-AntiSense 72 TTCTCTCTCAATCACGTTAAGGATGACGAACGTAAGCTGTGCTTACGATCATAGTGAACCTCTTCGAGGGAC dnaX Sense Primer 21 GCGTCAGGCGATATACGAAGA dnaX Antisense Primer 23 CAGACCGCCTTTACCAAACATAG dsbB-EGFP-Sense 87 GTGGTGATTTCCCAGCCGTTTAAAGCGAAAAAACGTGATCTGTTCGGTCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dsbB-EGFP-AntiSense 72 TAAATATAGCGGCAGGAAAAAAGCGCTCCCGCAGGAGCGCCGAATGGATTATAGTGAACCTCTTCGAGGGAC dsbB Sense Primer 21 ATCGCTTACCTGATTGTCGCA dsbB Antisense Primer 24 GCGAAGTTCTGATTTACTGAGGGT dsbC-EGFP-Sense 87 AAAGAGATGAAAGAATTCCTCGACGAACACCAAAAAATGACCAGCGGTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dsbC-EGFP-AntiSense 72 TTTCATCGACTTCACGGCGACGAAGTTGTATCTGTTGTTTCACGCGAATTATAGTGAACCTCTTCGAGGGAC dsbC Sense Primer 21 AGTTGTGCTGAGCAATGGCAC dsbC Antisense Primer 20 CAGCAAGGGAGGCAATTCAG duT-EGFP-Sense 87 TTCGACGCCACCGACCGCGGTGAAGGCGGCTTTGGTCACTCTGGTCGTCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG duT-EGFP-AntiSense 72 AACGAAATGTTTGCGGCTATGTTATGACGTTATTCGGATGCGTATGTGTTATAGTGAACCTCTTCGAGGGAC duT Sense Primer 24 GTTCCGGTAGTACAGGCTGAATTT duT Antisense Primer 23 TCTGCCATGTTACAAAATACCCC dxR-EGFP-Sense 87 GATGCGAACGCGCGTGAAGTCGCCAGAAAAGAGGTGATGCGTCTCGCAAGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG dxR-EGFP-AntiSense 72 GAAGCCCTACGCTAACAAATAGCGCGACTCTCTGTAGCCGGATTATCCTCATAGTGAACCTCTTCGAGGGAC dxR Sense Primer 21 CCACAATGTGTGGACGATGTG dxR AntiSense Primer 22 CGATCAGATGGCGCAGACTATA edA-EGFP-Sense 87 TACGACCGCATTACTAAGCTGGCGCGTGAAGCTGTAGAAGGCGCTAAGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG edA-EGFP-AntiSense 72 AAAAAAACGCTACAAAAATGCCCGATCCTCGATCGGGCATTTTGACTTTTATAGTGAACCTCTTCGAGGGAC edA Sense Primer 20 TGAAAAGCGTGCTGTGCATC edA Antisense Primer 22 GCCGGACAGGTAAAAGTACAGG efP-EGFP-Sense 87 GAAGTCATCAAAGTGGATACCCGCTCTGGTGAATACGTCTCTCGCGTGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG efP-EGFP-AntiSense 72 TGACCTGAATAAGTGATGGTGCAGCCTGCAGGCCGCACCACAACCGCATTATAGTGAACCTCTTCGAGGGAC efP Sense Primer 24 GTGGTTAAAGTTCCGCTGTTTGTA efP Antisense Primer 23 AACAAGAACGATAAGGCGTTTCA enO-EGFP-Sense 87 CTGGGCGAAAAAGCACCGTACAACGGTCGTAAAGAGATCAAAGGCCAGGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 174 enO-EGFP-AntiSense 72 AAAATGCCAGCCCGGAGGCTGGCATTTTTAAATCAGATAAAGTCAGTCTTATAGTGAACCTCTTCGAGGGAC enO Sense Primer 24 TACAACCAGCTGATTCGTATCGAA enO Antisense Primer 21 GTTCCGCTGGAGGCTCAGATA fabA-EGFP-Sense 87 TATACCGCCAGCGACCTGAAAGTCGGTCTGTTCCAGGATACGTCTGCCTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fabA-EGFP-AntiSense 72 CCTCCGCATTGCGGAGGTTTCGCCTTTTGATACTCTGTCTGATTATAATCATAGTGAACCTCTTCGAGGGAC fabA Sense Primer 20 ATGGCGAAGTGCTGGTTGAT fabA Antisense Primer 24 GGGTAGTAATGGCCTGATTCTGTC fabD-EGFP-Sense 87 ACCGCCTCGGCGCTGAACGAACCTTCAGCGATGGCAGCGGCGCTCGAGCTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fabD-EGFP-AntiSense 72 ACCGGTTACCAGTGCGATTTTTCCTTCAAAATTCATGATTTTCCTCTTTTATAGTGAACCTCTTCGAGGGAC fabD Sense Primer 20 CTGACGAAACGCATTGTCGA fabD Antisense Primer 20 AGTGCCAATAACTTTCGCGC fabG-EGFP-Sense 87 GCTTACATCACGGGTGAAACTTTGCATGTGAACGGCGGGATGTACATGGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fabG-EGFP-AntiSense 72 ATAACCACGCAGACTGCGCGCAACATGAGCATTTTGTGCAAATCGCGGTCATAGTGAACCTCTTCGAGGGAC fabG Sense Primer 18 TTGCATTCCTGGCATCCG fabG Antisense Primer 26 ATAACGCAAATCATTTTGCACTAATT fabH-EGFP-Sense 87 CTTGAAGCCTTTGGCGGTGGATTCACCTGGGGCTCCGCGCTGGTTCGTTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fabH-EGFP-AntiSense 72 CTGTCCAGGGAACACAAATGCAAATTGCGTCATGTTTTAATCCTTATCCTATAGTGAACCTCTTCGAGGGAC fabH Sense Primer 20 TGGATCGCCACGGTAATACC fabH Antisense Primer 21 ATATCAGCCAGCATTCCAACG fabZ-EGFP-Sense 87 GGTAAAGTAGTTTGCGAAGCAACGATGATGTGTGCTCGTAGCCGGGAGGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fabZ-EGFP-AntiSense 72 TTCCACAATGGCGGTTGGATGCACAAAGGCGGATTTATCAATCACGTATCATAGTGAACCTCTTCGAGGGAC fabZ Sense Primer 20 ACCCGTTTTAAAGGGGTTGC fabZ Antisense Primer 23 CCAACGATACAAAAAGGACCAAT fadD-EGFP-Sense 87 TTGCGACGAGAATTACGTGACGAAGCGCGCGGCAAAGTGGACAATAAAGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fadD-EGFP-AntiSense 72 GTCAAAAAAAACGCCGGATTAACCGGCGTCTGACGACTGACTTAACGCTCATAGTGAACCTCTTCGAGGGAC fadD Sense Primer 24 GAGTTTCGTGATGAGTTACCGAAA fadD Antisense Primer 22 CATCGTCCGTGGTAATCATTTG ffH-EGFP-Sense 87 AAGATGATGAGAAGCATGAAGGGTATGATGCCCCCAGGCTTCCCTGGTCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 175 ffH-EGFP-Antisense 72 GGATGACGAGCACATCCCGGTGCCAAAATGGCAAACAAGCCAGGCCGATTATAGTGAACCTCTTCGAGGGAC ffH Sense Primer 22 GCGCATGATGAAGAAAATGAAG ffH Antisense Primer 19 CCGGAGCGTACACGCAGTA fiS-EGFP-Sense 87 GGCATCAACCGTGGTACGCTGCGTAAAAAATTGAAAAAATACGGCATGAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fiS-EGFP-AntiSense 72 CCCCATGCCGAGTAGCGCCTTTTTAATCAAGCATTTAGCTAACCTGAATTATAGTGAACCTCTTCGAGGGAC fiS Sense Primer 21 TGCAATACACCCGTGGTAACC fiS Antisense Primer 23 GGTCACTCCCTTTGTGACACCTA fmT-EGFP-Sense 87 GACCTCCTGAACTCTCGTCGGGAATGGTTTGTTCCGGGCAACCGTCTGGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fmT-EGFP-AntiSense 72 CATAAGTATAAAAACGCCCGGCAAGACCGGGCTTAGAAGAGTGGACTATCATAGTGAACCTCTTCGAGGGAC fmT Sense Primer 22 ACCTGCTCTCGTTACAACCTGC fmT Antisense Primer 21 CCTTGCTCGACGACTTGTTCA folA-EGFP-Sense 87 GCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG folA-EGFP-AntiSense 72 GACGCGACCGGCGTCGCATCCGGCGCTAGCCGTAAATTCTATACAAAATTATAGTGAACCTCTTCGAGGGAC folA Sense Primer 23 GATGACTGGGAATCGGTATTCAG folA Antisense Primer 22 CACAGCCTGATATAGGAAGGCC folB-EGFP-Sense 87 GCGAATGTTGGCGTAATCATTGAGCGTGGCAATAATCTGAAAGAAAATAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG folB-EGFP-AntiSense 72 CTACTGCGTATGACCAGGTTATAACCGTTTGGTTTAACAGCTGTAAAATTATAGTGAACCTCTTCGAGGGAC folB Sense Primer 22 GTGCGTATCAAACTCAGCAAGC folB Antisense Primer 24 TTATTAAAATGTACCGCTTGTCCG folC-EGFP-Sense 87 ACGGTCGCACATGTCATGGAAGTGATTGACGCGAGGAGAAGCGGTGGCAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG folC-EGFP-AntiSense 72 ACCCCCAGCGCCACCAGCACGATCGTGCCCACTAACCGATTCTGAAACTTATAGTGAACCTCTTCGAGGGAC folC Sense Primer 19 AAAGCGGAAGACACCGTGC folC Antisense Primer 20 CCAGCAGCCCTGGAAGTACA folD-EGFP-Sense 87 ATTGAAAACACGCTACAGGCGTGCGTTGAATATCATGATCCACAGGATGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG folD-EGFP-AntiSense 72 TCGCACAGCTCAACGTGCGGATGTTTACCTAAAGAAAATGTCGCCATGTTATAGTGAACCTCTTCGAGGGAC folD Sense Primer 23 GCTAAACGCGCCTCATACATTAC folD Antisense Primer 19 TCGCTCCAGCCTTCCAGTT folE-EGFP-Sense 87 TCCAGTCAGAATACGCGCCACGAGTTTCTGCGCGCTGTGCGTCATCACAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 176 folE-EGFP-AntiSense 72 CGAACAAAATCGAGCGTGACGTTGCGCTCCATGGTTCCTGCCTTTTAATCATAGTGAACCTCTTCGAGGGAC folE Sense Primer 19 ACCAGTGCCACGACAACGA folE Antisense Primer 21 ATAGCAGGATCCCCAGAATGG folK-EGFP-Sense 87 ATGTTGCGTCAAATCTTACATACAAGAGCATTTGACAAATTAAACAAATGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG folK-EGFP-AntiSense 72 GATGAGATATGATTTTCAGAAAATATTTATATTCGCAATATAAATAAATTATAGTGAACCTCTTCGAGGGAC folK Sense Primer 20 ATTTATGCTGTGGCCGCTGT folK Antisense Primer 23 GGTTAGGGTATGGAGATGGGATT folP-EGFP-Sense 87 ATGCGGGTGGTGGAAGCCACTCTGTCTGCAAAGGAAAACAAACGCTATGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG folP-EGFP-AntiSense 72 GCATCCCCTACACGACCACGAATCCCATCGGTACCGAAATATTTACGATTATAGTGAACCTCTTCGAGGGAC folP Sense Primer 23 ATCATTCGTGTTCATGACGTCAA folP Antisense Primer 22 AGCACAAAATCAGGTGTGATCG frR-EGFP-Sense 87 AAGAAAATTGAAGCGGCGCTGGCAGACAAAGAAGCAGAACTGATGCAGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG frR-EGFP-AntiSense 72 AGCCGATCCGCAAGGATCTACTGAGCGGCGTTTTTGTCGTTCAAGAAATCATAGTGAACCTCTTCGAGGGAC frR Sense Primer 22 GAGATCAGCGAAGACGACGATC frR Antisense Primer 23 CAGAATGGTGAGTTGCTTCATGA ftsA-EGFP-Sense 87 TCAGTTGGCTCGTGGATCAAGCGACTCAATAGTTGGCTGCGAAAAGAGTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ftsA-EGFP-AntiSense 72 ATTTGTGCCTGTCGCCTGAGGCCGTAATCATCGTCGGCCTCATAAAAATTATAGTGAACCTCTTCGAGGGAC ftsA Sense Primer 25 AACGGTGAAGCTGAAGTAGAAAAAC ftsA Antisense Primer 21 CTTTAATCACCGCGTCATTGG ftsE-EGFP-Sense 87 CGCATGCTCACCCTGAGCGATGGTCACTTGCATGGAGGCGTGGGCCATGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ftsE-EGFP-AntiSense 72 CGATCAAGACGCCCGCCAAACTGCCGAATATGATTGATTGCATCGCGCTTATAGTGAACCTCTTCGAGGGAC ftsE Sense Primer 20 AACCGTATTGATGGCAACGC ftsE Antisense Primer 22 AACGTTGGTTTTGCGATTTACC ftsI-EGFP-Sense 87 GATAAAAATGAATTTGTGATTAATCAAGGCGAGGGGACAGGTGGCAGATCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ftsI-EGFP-AntiSense 72 GCTCGCGAAGGTGCGTCTGGCACCCACGGAGCAAGAAGGTCGCGCAAATTATAGTGAACCTCTTCGAGGGAC ftsI Sense Primer 22 GCGTATTGCGTACCATGAACAT ftsI Antisense Primer 21 CGGCTGTCGAGTGTCATCTCT ftsJ-EGFP-Sense 87 TCTCGTGCACGTTCGCGGGAAGTGTATATTGTAGCGACCGGGCGTAAACCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 177 ftsJ-EGFP-AntiSense 72 GATACTCTATATCCAGCATCTTTCAAACTTTCGTCTGAAATCTCCCGGTTATAGTGAACCTCTTCGAGGGAC ftsJ Sense Primer 21 AGGTCAAAGTTCGTAAGCCGG ftsJ Antisense Primer 19 GCACAACGGCAATGACCAG ftsW-EGFP-Sense 87 GATTATGAAACGCGTCTGGAGAAAGCGCAGGCGTTTGTACGAGGTTCACGACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ftsW-EGFP-AntiSense 72 GTCCACCGGTTCCGCCTGCCATCACCATTAATCGCTTTCCTTGACCACTCATAGTGAACCTCTTCGAGGGAC ftsW Sense Primer 24 ACTGATTATGTCGACAGCCATCAT ftsW Antisense Primer 20 TGCCAACCCTGAGCCATTAG ftsX-EGFP-Sense 87 GTGGCAGCGTGGCTTGCCACGGTACAACATTTACGCCACTTTACGCCTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ftsX-EGFP-AntiSense 72 TTCCCTGCTACGGAACCCATTGCAGGGAAAGAGTATAACACGCTTTTATTATAGTGAACCTCTTCGAGGGAC ftsX Sense Primer 22 TATTGCTGCTGGTATGCTCGAT ftsX Antisense Primer 22 GAAATACCGGGAAGAGACAACG fumC-EGFP-Sense 87 GACAGCTGGGTACGGCCAGAACAGATGGTCGGCAGTATGAAAGCCGGGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fumC-EGFP-AntiSense 72 GCAGCCGCTTCGTTTGATCATTCCACGGCTGCACCTGTATGTTGCAGATTATAGTGAACCTCTTCGAGGGAC fumC Sense Primer 20 CGCTGGGGTATCTTAGCGAA fumC Antisense Primer 23 GACAATGTGCAGCACCGTTATAA fuR-EGFP-Sense 87 CACTGTGCCGAAGGCGATTGCCGCGAAGATGAACATGCGCACGAAGGCAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fuR-EGFP-AntiSense 72 TGCATAAAAAAGCCAACCCGCAGGTTGGCTTTTCTCGTTCAGGCTGGCTTATAGTGAACCTCTTCGAGGGAC fuR Sense Primer 20 TGGCATTCGCCTGACTAACC fuR Antisense Primer 26 AAATAAGTGAGAGCTGTAACTCTCGC fusA-EGFP-Sense 87 GATGAAGCGCCGAGTAACGTTGCTCAGGCCGTAATTGAAGCCCGTGGTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG fusA-EGFP-AntiSense 72 CTCTCCCCTTCAGGAGAGAGCACGGGACTTTGGTATTAACCCTTAGGCTTATAGTGAACCTCTTCGAGGGAC fusA Sense Primer 21 TCTGACCAAAGGTCGTGCATC fusA Antisense Primer 22 TAACGTGCGGTTTTGTACGTTC galE-EGFP-Sense 87 GCGCAGGACACCTGGCACTGGCAGTCACGCCATCCACAGGGATATCCCGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG galE-EGFP-AntiSense 72 GCGGCGATGTGGATGATCAACGGGATTAAATTGCGTCATGGTCGTTCCTTATAGTGAACCTCTTCGAGGGAC galE Sense Primer 20 GTAACGCGCACACTCGATGA galE Antisense Primer 19 AGAATCCATTGCCCGGTGA galU Antisense Primer 21 CGGGATTTTTTATTGTCCGGT 178 galU-EGFP-AntiSense 72 CGGCGTCGATTGCTCAACGCCGTTTCGTGGATAACACCGATACGGATGTTATAGTGAACCTCTTCGAGGGAC galU-EGFP-Sense 87 CTTGGCACGGAATTTAAAGCCTGGCTTGAAGAAGAGATGGGCATTAAGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG galU Sense Primer 20 GCAGGCCTTCGTTGAATACG gcpE-EGFP-Sense 87 GCCAGTCAGCTGGACGAAGCGCGTCGAATTGACGTTCAGCAGGTTGAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gcpE-EGFP-AntiSense 72 ATGCGGGTTCAATCATACACGGGAAGCGAGGCGCTTCCCATCACGTTATTATAGTGAACCTCTTCGAGGGAC gcpE Sense Primer 22 CAACGATATGATCGACCAGCTG gcpE Antisense Primer 87 GCCAGTCAGCTGGACGAAGCGCGTCGAATTGACGTTCAGCAGGTTGAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gcvA-EGFP-Sense 87 CTGGCGAAAGCCGCTGCTGAACAAGAAAAATTCCGCTTTCGTTATGAACAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gcvA-EGFP-AntiSense 72 GGCGAAAATCAGCATAAAACGGCTGGTCATGGTCGTACCCTACGTAAATTATAGTGAACCTCTTCGAGGGAC gcvA Sense Primer 22 CATGACAGTCAGGCAGAACTGG gcvA Antisense Primer 21 CCACAAAAATGAAGCCGCTAA gcvH-EGFP-Sense 87 CTGGAATCACTGCTGGATGCGACCGCATACGAAGCATTGTTAGAAGACGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gcvH-EGFP-AntiSense 72 TAAATTTTCCTCACCCTGATCCTCTCCCGCAGAAGAGGAATAAAGCCGTTATAGTGAACCTCTTCGAGGGAC gcvH Sense Primer 22 TAAAATCAAAGCCAGCGATGAA gcvH Antisense Primer 24 CTCTCCTTACGAAGAGAGTGAGGG gidA-EGFP-Sense 87 TCCATTCTGCTGGTGTGGCTGAAAAAACAGGGTATGCTGCGTCGTAGCGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gidA-EGFP-AntiSense 72 TTGATGCGTTGCCTGGTAAGCGGGTGCTTACCAGGCATTTTTAATGCGTTATAGTGAACCTCTTCGAGGGAC gidA Sense Primer 19 GGCCAAGCTTCGCGTATTT gidA Antisense Primer 23 GTTTGTTGAGCACGGTGATTACC gidB-EGFP-Sense 87 CCAGCCCTGGATGGCGAACGTCATCTGGTGGTGATTAAAGCAAATAAAATTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gidB-EGFP-AntiSense 72 TTGTTAACAGTCTAACCGGTCAATTTTTTATGATTTTTTTGATAAAAATTATAGTGAACCTCTTCGAGGGAC gidB Sense Primer 25 CAGGTCGAATCAGTGGTTAAACTTC gidB Antisense Primer 21 CAAAAGAGTGAACGTGGCGTT glmM-EGFP-Sense 87 GCGCAGGTGACTGAATTTGCACACCGCATCGCCGATGCAGTAAAAGCCGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glmM-EGFP-AntiSense 72 GAGAAAAAAGGCGACAATCGCCGCCTTTTTAGCCAGTTATCTAACGCTTTATAGTGAACCTCTTCGAGGGAC glmM Sense Primer 20 GTGATGGTGGAAGGCGAAGA glmM Antisense Primer 21 ACAGCAAAACCGATGTGTTCG glmS-EGFP-Sense 87 GGCACCGACGTTGACCAGCCGCGTAACCTGGCAAAATCGGTTACGGTTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 179 glmS-EGFP-AntiSense 72 AAACATAACAGGAAGAAAAATGCCCCGCTTACGCAGGGCATCCATTTATTATAGTGAACCTCTTCGAGGGAC glmS Sense Primer 21 TTGCACCGATCTTCTACACCG glmS Antisense Primer 22 ACATGGAGTTGGCAGGATGTTT glmU-EGFP-Sense 87 CGTGTGCCGCAGACTCAGAAAGAAGGCTGGCGTCGTCCGGTAAAGAAAAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glmU-EGFP-AntiSense 72 AGATTATGTTATCTCCTCATCCCATGTGACCGGGTTAGCCGGCCAGAATCATAGTGAACCTCTTCGAGGGAC glmU Sense Primer 23 GGCGAAAATGCATTAGCTATCAG glmU Antisense Primer 25 TTTATAGTTACTGCTTGTGGGAGGG glnA-EGFP-Sense 87 CGCGTGCGTATGACTCCGCATCCGGTAGAGTTTGAGCTGTACTACAGCGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glnA-EGFP-AntiSense 72 CCAGGCCTGCCAGAGACAGGCGAAAAGTTTCCACGGCAACTAAAACACTTATAGTGAACCTCTTCGAGGGAC glnA Sense Primer 22 CGAAGCAATTGATGCGTACATC glnA Antisense Primer 19 GGCCGGATAAGACGCATTT glnD-EGFP-Sense 87 CAGGAAGTGCATCAGCGGTTGACAGAGGCCCTCAATCCAAACGATAAAGGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glnD-EGFP-AntiSense 72 GTTCTGTAACTGCTGCATTGTTAAACTCTTTTCATATCAGTAAACACATCATAGTGAACCTCTTCGAGGGAC glnD Sense Primer 22 TTATTCATAATTGCCACCGCTG glnD Antisense Primer 19 GGCGTTCAAAAGCGGTTTC glnE-EGFP-Sense 87 GCAGAGCGTGAACTGGTGCGGGCAAGCTGGCAGAAGTGGCTGGTGGAAGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glnE-EGFP-AntiSense 72 TTCCTGTCTCCTGAGAGATTCAAAATTTGCGCGCGATAATACCATACTTCATAGTGAACCTCTTCGAGGGAC glnE Sense Primer 22 ATCTGGCATTACAGGAATTGCC glnE Antisense Primer 20 ACGTTCAAACTCTGGCAGCG glnS-EGFP-Sense 87 GTATTTAACCGCACCGTTGGGCTGCGTGATACCTGGGCGAAAGTAGGCGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glnS-EGFP-AntiSense 72 TTGCCGGATGCGACGTAAACGCCCCATCCGGCATAGCGAAACTTAAAATTATAGTGAACCTCTTCGAGGGAC glnS Sense Primer 22 GTTACTTCTGCCTCGATAGCCG glnS Antisense Primer 20 GCCTGATAAGCGTAGCGCAT gloA-EGFP-Sense 87 TACAAAATTGAGTTAATCGAAGAGAAAGACGCCGGTCGCGGTCTGGGCAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gloA-EGFP-AntiSense 72 TCGATGCAGTAAAGATGCGGGCGCGATGAGTTCACGCCCGGCAGGAGATTATAGTGAACCTCTTCGAGGGAC gloA Sense Primer 21 CTACGGTTATCGCGTTTGTGG gloA Antisense Primer 21 ATAAATTGCAGCGCGCATTAT gloB-EGFP-Sense 87 CAACAACCTGAAGAGCGTTTTGCATGGTTAAGGTCAAAGAAAGATAGGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 180 gloB-EGFP-AntiSense 72 AGACGACCGATCATAACGGCGAACGGAGCCGATGACAAGAAAGTTTTATCATAGTGAACCTCTTCGAGGGAC gloB Sense Primer 21 TCTGAAAAATGAGCGGCAAAT gloB Antisense Primer 27 TTCATGTGTGTGTCAATAGTTGCTTAA glpK-EGFP-Sense 87 GGCTGGAAAAAAGCGGTTAAACGCGCGATGGCGTGGGAAGAACACGACGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glpK-EGFP-AntiSense 72 GTTTCGGGACTACCGGATGCGGCATAAACGCTTCATTCGGCATTTACATTATAGTGAACCTCTTCGAGGGAC glpK Sense Primer 20 GCATCGAAACCACTGAGCGT glpK Antisense Primer 20 TATTGATGTGTGCGGGGTTG gltA-EGFP-AntiSense 72 CGCCATATGAACGGCGGGTTAAAATATTTACAACTTAGCAATCAACCATTATAGTGAACCTCTTCGAGGGAC gltA-EGFP-Sense 87 CAGCTGTATACAGGATATGAAAAACGCGACTTTAAAAGCGATATCAAGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gltA Sense Primer 22 AGTGACGGTATGAAGATTGCCC gltA Antisense Primer 26 GGTTATAAATGCGACTACCATGAAGT gltX-EGFP-Sense 87 GAGCGTATCAACAAAGCGCTGGATTTTATTGCTGAACGCGAAAATCAGCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gltX-EGFP-AntiSense 72 AAAAACGGCAGGATATTATCTCCTGCCGTTTATCTTTTTACACGCTAATTATAGTGAACCTCTTCGAGGGAC gltX Sense Primer 22 GTTACCGTTCACGCAATTGGTA gltX Antisense Primer 24 GTCTCGATATTGACGAATCAGCAT glyA-EGFP-Sense 87 CGCATCAAAGGTAAAGTTCTCGACATCTGCGCACGTTACCCGGTTTACGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glyA-EGFP-AntiSense 72 GCATCCGGCATGAACAACGAGCACATTGACAGCAAATCACCGTTTCGCTTATAGTGAACCTCTTCGAGGGAC glyA Sense Primer 22 GCTGGACAGCATCAATGATGAA glyA Antisense Primer 21 GTTTTGTAGGCCGGATAAGGC glyQ-EGFP-Sense 87 TACGCTTCCCGTGAAGCCCTCGGCTTCCCGATGTGCAACAAAGATAAGTAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glyQ-EGFP-AntiSense 72 TTCAGTGCCGATTTCCACCAGAAAAGTTTTCTCAGACATAGCCGCCTCTTATAGTGAACCTCTTCGAGGGAC glyQ Sense Primer 21 GAGCGTCAGCGCTACATTCTG glyQ Antisense Primer 21 AAGTTCGCAGCAAAGGACTCA glyS-EGFP-Sense 87 GAGAAACTGCGCGAACTGTTCCTGCGCGTTGCGGATATTTCGCTGTTGCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG glyS-EGFP-AntiSense 72 GCGATAAAAAAAGCCTGCCAGATGGCAGGCTATTTAATAACGGCGTTATTATAGTGAACCTCTTCGAGGGAC glyS Sense Primer 21 TTGCGTATCAACCGTCTGACC glyS Antisense Primer 21 CAAGCACCCGTGGCTATTCTA gmK-EGFP-Sense 87 AGCCGCCAAAAGCAGCGTCATGACGCTTTAATCAGCAAATTGTTGGCAGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 181 gmK-EGFP-AntiSense 72 AAAGCTCCACAGGTGAAGAAATGACTGGGCATGATACTGAAATCAGGTTCATAGTGAACCTCTTCGAGGGAC gmK Sense Primer 19 ACCATTATTCGCGCCGAAC gmK Antisense Primer 20 CTGAACAGTTACGCGTGCCA goR-EGFP-Sense 87 AATACCGTCGCCATTCACCCAACGGCGGCAGAAGAGTTCGTGACAATGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG goR-EGFP-AntiSense 72 GAAACGTAATTAAGGGCTAAGAGCACACTACTCTTAGCCCTTTAACATTTATAGTGAACCTCTTCGAGGGAC goR Sense Primer 22 GGCAACCAAAAAAGACTTCGAC goR Antisense Primer 24 TTCTGCTTCAGCTTCTGAACTGAT gpH-EGFP-Sense 87 CTGCCCGCATTAGGGCTTCCGCATAGCGAAAATCAGGAATCGAAAAATGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gpH-EGFP-AntiSense 72 CCAATGGTCAATTCACCTGAGGGCTGTGCGCCACTAAAAACGATGGGCTTATAGTGAACCTCTTCGAGGGAC gpH Sense Primer 25 AGCCAGCCTGATGTAATTTATCAGT gpH Antisense Primer 24 TGGTAGTCATCCTGCATGTTTACC gpsA-EGFP-Sense 87 GCGCGCGAGGCAGCATTGACTTTACTAGGTCGTGCACGCAAGGACGAGCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gpsA-EGFP-AntiSense 72 CCCGTTCTGCGCGGGCCGGGTCATAGCGGTAACAAAGGTTCCCTGGGGTTATAGTGAACCTCTTCGAGGGAC gpsA Sense Primer 21 TTGAAATGCCAATAACCGAGG gpsA Antisense Primer 25 GCTTACTCCACACGATGAGATAATG greA-EGFP-Sense 87 AAAACGCCGGGCGGCGAAGTAGAATTTGAAGTAATTAAGGTGGAATACCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG greA-EGFP-AntiSense 72 TTTTTCCTTTCTTTACAATACATCAACATCTTGAGTATTGGGTAATTCTTATAGTGAACCTCTTCGAGGGAC greA Sense Primer 24 GCAAAGAAGAAGATGATGTTGTGG greA Antisense Primer 21 TCGTTGATAAAAGGCCGCATA groL-EGFP-Sense 87 TTAGGCGCTGCTGGCGGTATGGGCGGCATGGGTGGCATGGGCGGCATGATGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG groL-EGFP-AntiSense 72 AGACATTTCTGCCCGGGGGTTTGTTTATTTCTGCGAGGTGCAGGGCAATTATAGTGAACCTCTTCGAGGGAC groL Sense Primer 20 GTGGCTGGCCTGATGATCAC groL Antisense Primer 23 CGTCGTCCGTGTCTGAATCTTAT groS-EGFP-Sense 87 GAAGAAGTGTTGATCATGTCCGAAAGCGACATTCTGGCAATTGTTGAAGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG groS-EGFP-AntiSense 72 GCCATTATCTTTATTCCTTAAATTCGTATGTTCAGTGTCGTGCGCGGATTATAGTGAACCTCTTCGAGGGAC groS Sense Primer 23 GGCTACGGTGTGAAATCTGAGAA groS Antisense Primer 20 CACGAGCGTCGTTACCGAAT grpE-EGFP-Sense 87 AATGGTCGTACGATTCGTGCGGCGATGGTTACTGTAGCGAAAGCAAAAGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 182 grpE-EGFP-AntiSense 72 CGGCCCGGCATTCGCATGCAGGGCCGTGAATTATTACGAAAGCAGAAATTATAGTGAACCTCTTCGAGGGAC grpE Sense Primer 23 ACGTACTGGGCATTATGCAGAAG grpE Antisense Primer 22 AGTTTTTCCTGTGAAACCGCTG gshB-EGFP-Sense 87 TCGATCACCGGAATGTTAATGGATGCCATCGAAGCACGTTTACAGCAGCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gshB-EGFP-AntiSense 72 GGGCGCTGTCACTCAGAGTCTCAACGAGATCCTTCTCGCTAAGGTGGGTTATAGTGAACCTCTTCGAGGGAC gshB Sense Primer 24 CCTGTATTCGTGAGATTGAAGCAG gshB Antisense Primer 18 AACAGCGCCCAGTATGCG guaA-EGFP-Sense 87 CGCGTGGTGTATGACATCAGCGGCAAGCCGCCAGCTACCATTGAGTGGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG guaA-EGFP-AntiSense 72 CCCTCTGTAGTAACAGAGGGTTTTGTTCATTCATAGTGCAGGGTCAAATCATAGTGAACCTCTTCGAGGGAC guaA Sense Primer 19 ATTTCCTCGGTCGCGTTTC guaA Antisense Primer 26 GACAGCTGGTTTAATTAATCGATGTT guaB-EGFP-Sense 87 GTTCACGACGTGACCATTACTAAAGAGTCCCCGAACTACCGTCTGGGCTCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG guaB-EGFP-AntiSense 72 GTGAAACAGATAATATAAATCGCCCGACATGAAGTCGGGCGAAGAGAATCATAGTGAACCTCTTCGAGGGAC guaB Sense Primer 21 CGAACTGCGTACTAAAGCGGA guaB Antisense Primer 21 TCCGTCATTGACGCTTATTCC gyrA-EGFP-Sense 87 GACGATGAAATCGCTCCGGAAGTGGACGTTGACGACGAGCCAGAAGAAGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gyrA-EGFP-AntiSense 72 TCAATTCAAACAAGGGAGATAGCTCCCTTTTGGCATGAAGAAGTAAAATTATAGTGAACCTCTTCGAGGGAC gyrA Sense Primer 22 AAGATCTGGATACCATCGACGG gyrA Antisense Primer 24 AACTTTACCGTGCCCTAATACGAC gyrB-EGFP-Sense 87 CGCCGTGCGTTTATTGAAGAGAACGCCCTGAAAGCGGCGAATATCGATATTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG gyrB-EGFP-AntiSense 72 AGGCCTGATAAGCGTAGCGCATCAGGCACGCTCGCATGGTTAGCGCCATTATAGTGAACCTCTTCGAGGGAC gyrB Sense Primer 19 TGCTGCCGACCAGTTGTTC gyrB Antisense Primer 27 ATTCAATACATTGCAAGATTTTCGTAG hemA-EGFP-Sense 87 GACGGGGATAACGAACGCCTGAATATTCTGCGCGACAGCCTCGGGCTGGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hemA-EGFP-AntiSense 72 GCTTCATAGGCGTAAATGCACCCTGTAAAAAAAGAAAATGATGTACTGCTATAGTGAACCTCTTCGAGGGAC hemA Sense Primer 20 ATGCGCCAACGAAATCACTT hemA Antisense Primer 19 GGGCTTCCAGTTTGGCAAC hemB-EGFP-Sense 87 CTGATTTTCAGCTACTTTGCGCTGGATTTGGCTGAGAAGAAGATTCTGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 183 hemB-EGFP-AntiSense 72 CACCGTACTTTCAACAGGTTAACTCCCCCTTTCTGAGAGGAAACAAAATTATAGTGAACCTCTTCGAGGGAC hemB Sense Primer 22 GTCGTGCTCGAAAGCTTAGGTT hemB Antisense Primer 22 GATCCTTGGGGATAAACCGTTT hemC-EGFP-Sense 87 AACGGCGCGCGCGAGATCCTCGCTGAAGTCTATAACGGAGACGCCCCGGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hemC-EGFP-AntiSense 72 GGCTCACTAACTCTTCTCCAGCGGGAGACGGGCGGGTGACAAGGATACTCATAGTGAACCTCTTCGAGGGAC hemC Sense Primer 20 GATTTCGCTGGCAGAAGAGC hemC Antisense Primer 20 CTCAATCAGCGGAAAATGCC hemE-EGFP-Sense 87 GCTGGCGTGTTCGTGGAGGCAGTGCATCGACTGTCTGAACAGTATCACCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hemE-EGFP-AntiSense 72 CAGTTCGATTTGTTGAGCGCGTAATGACGCGAGATCCATAATCACTCCTTATAGTGAACCTCTTCGAGGGAC hemE Sense Primer 20 TTGGTCACGGCATTCATCAG hemE antisense Primer 21 ATATTTCAGCAGCACCATCGC hemF-EGFP-Sense 87 GGCAGCCCAGAAGCGGCGTTAAGTGAGTTTATTAAGGTCAGGGATTGGGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hemF-EGFP-AntiSense 72 GCCGTCACTGACGCTGCATCAGCGGATGCGGGAGTGGGGGTGAGGGAGTTATAGTGAACCTCTTCGAGGGAC hemF Sense Primer 24 TACGCTGGGAATATGATTATCAGC hemFAntisense Primer 21 CACGGATTACCAGCAGCTGTT hemH-EGFP-Sense 87 GCCACGCCGGAACATATCGAAATGATGGCTAATCTTGTTGCCGCGTATCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hemH-EGFP-AntiSense 72 CGCCGACGAGGCTCTTCGCGACGGCGCTCAGTTCTTTACCGCTCAGCTTTATAGTGAACCTCTTCGAGGGAC hemH Sense Primer 21 GCAAAACCGTGAGGTCTTCCT hemH Antisense Primer 23 TTTGCATTATTCACGGATGATGA hemK-EGFP-Sense 87 TGCCGTGACTATGGTGATAACGAGCGCGTAACGCTCGGCCGCTATTATCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hemK-EGFP-AntiSense 72 AAAGCGCGATACTAATAAGATGAACACTAAGCAGTGTAGAAAAACTTGTCATAGTGAACCTCTTCGAGGGAC hemK Sense Primer 20 ACAAGCATTTATCCTCGCGG hemK Antisense Primer 24 GCCAGAAACGTAAGGTTAATAGCC hemL-EGFP-Sense 87 GAAGATATCAATAACACCATCGATGCTGCACGTCGGGTGTTTGCGAAGTTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hemL-EGFP-AntiSense 72 CGTAGGTCGGATAAGGCGTTCACGCCGCATCCGACAAACCATGCTGGATCATAGTGAACCTCTTCGAGGGAC hemL Sense Primer 22 ATGCTGGACGAAGGTGTTTACC hemL Antisense Primer 20 GCGTCTTATCGGGCCTACAA hflB-EGFP-Sense 87 CCGCGTACGCCGAACCCGGGTAACACCATGTCAGAGCAGTTAGGCGACAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 184 hflB-EGFP-AntiSense 72 GCACGCCCCGGGGTTTTCGGTACAAATACAGTCATCTGATGCGGGAACTTATAGTGAACCTCTTCGAGGGAC hflB Sense Primer 23 ACAATTCTGGCGACAATGGTAGT hflB Antisense Primer 22 AGTGAAGTACCCTGGGCAAAGA hflC-EGFP-Sense 87 CCGGATAGCGATTTCTTCCGCTACATGAAGACGCCGACTTCCGCAACGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hflC-EGFP-AntiSense 72 ATCCCTGAGGATGCGGTGGCTTTATTGACCTGTACCGCAGTCGTTATATTATAGTGAACCTCTTCGAGGGAC hflC Sense Primer 23 AATCAGGACGTGATGGTCATGAG hflC Antisense Primer 23 AGCCAGATTGTCGAATTCATTCA hflK-EGFP-Sense 87 GACCAACGCCGCGCCAACGCGCAGCGTAACGACTACCAGCGTCAGGGGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hflK-EGFP-AntiSense 72 GCACTACCAGCACGATGATGATAATCGCGATAACTGACTTACGCATCGTTATAGTGAACCTCTTCGAGGGAC hflK Sense Primer 21 AACCAGTGGAGCAAGCAACAC hflK Antisense Primer 20 GCGCTCACCTTCTTTGACGA hflX-EGFP-Sense 87 GACTGGCGTCGCCTCTGTAAACAAGAACCGGCGTTGATCGATTACCTGATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hflX-EGFP-AntiSense 72 GTCTTTCGACGCCAGAGGATATGACTCCACGCTTCAGACGCTACGCCGTTATAGTGAACCTCTTCGAGGGAC hflX Sense Primer 21 TCTGCAAGTTCGTATGCCGAT hflX Antisense Primer 23 CATATTTGTTATGCGGTGATCCC hfQ-EGFP-Sense 87 GGTAGCAGCGCGCAGAATACTTCCGCGCAACAGGACAGCGAAGAAACCGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hfQ-EGFP-AntiSense 72 GGGAACGCAGGATCGCTGGCTCCCCGTGTAAAAAAACAGCCCGAAACCTTATAGTGAACCTCTTCGAGGGAC hfQ Sense Primer 24 GGTTTCTCATCACAGTAACAACGC hfQ Antisense Primer 26 CGTATAACCCTCTAAATAGATCAGCG hisA-EGFP-Sense 87 CTGGAAGGTAAATTCACCGTGAAGGAGGCCATCGCATGCTGGCAAAACGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hisA-EGFP-AntiSense 72 ACTGTACGCCTTTCACCACCTGACCATCACGAACGTCGAGACATGGGATTATAGTGAACCTCTTCGAGGGAC hisA Sense Primer 20 GCGTAATAGTTGGTCGGGCA hisA Antisense Primer 22 CGATATCGCCAATGATTTCATG hisD-EGFP-Sense 87 GCCCACAAAAATGCCGTTACTTTGCGTGTTAACGCCCTTAAGGAGCAAGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hisD-EGFP-AntiSense 72 TCAGGTTGCGGACGTTTTCACGCGCTAAATCGGTAATAGTCACGGTGCTCATAGTGAACCTCTTCGAGGGAC hisD Sense Primer 24 CTGGCTTCAACCATAGAAACACTG hisD Antisense Primer 20 GTTGGCGTTCAGCCAGACAT hisF-EGFP-Sense 87 GGTGAATTAAAAGCGTACCTGGCAACACAGGGCGTGGAGATCAGGATATGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 185 hisF-EGFP-AntiSense 72 TAAGTCCGTCGGTTTTTTCCCAGTCCAGTTCGCGACGTTGTTGTTCTGTTATAGTGAACCTCTTCGAGGGAC hisF Sense Primer 21 GCAGCTTCCGTATTCCACAAA hisF Antisense Primer 21 GATACCGCGTGTTGCACAATC hisS-EGFP-Sense 87 ACGGCAGTTGCGCAGGATAGCGTAGCCGCGCATTTGCGCACGTTACTGGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hisS-EGFP-AntiSense 72 CCTGGTCGTTTTCGTTCTCGTAAATTTCCACGCTGTCCTTCTCCTTCCTTATAGTGAACCTCTTCGAGGGAC hisS Sense Primer 20 GAAGGATTTGCGCTCTGGTG hisS Antisense Primer 20 AACAGCCAGTGCTTTGCCAT holA-EGFP-Sense 87 TTATCTCTTCTGTTGTGCCATAAACCCCTGGCGGACGTATTTATCGACGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG holA-EGFP-AntiSense 72 TGCACCGGATCAAAGGTGCCGCCAAACAGAGCCTGTAAAGATTTCATATCATAGTGAACCTCTTCGAGGGAC holA Sense Primer 23 ACCCTCAAACAAGATTACGGTCA holA Antisense Primer 22 TTTCCACGGGTTTTAGATGACC holB-EGFP-Sense 87 CGTATTGAGCATTACCTGCAACCGGGCGTTGTGCTACCGGTTCCTCATCTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG holB-EGFP-AntiSense 72 AGACCATCGAGATGGCAGTGTGAGTCGACTAAAAACATGATGTCTCTCTTATAGTGAACCTCTTCGAGGGAC holB Sense Primer 20 GCGAGCTTCTCATCACCGAT holB Antisense Primer 23 TCCACGTCCTTATGCAAAGATTC hrpA-EGFP-Sense 87 TATCCGATTTCAGATAAGCGTATTTTGCAGGCGATGGAGCAGATTAGCGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hrpA-EGFP-AntiSense 72 GGCGCTAAATGCTTACCGGGTTTTTTCTTTATCAGGCAAATAGCAGGGTTATAGTGAACCTCTTCGAGGGAC hrpA Sense Primer 21 TGCGCGTTAGTTACTTCGCTC hrpA Antisense Primer 21 CACTCCGCAAGCCTGACATAT hslU-EGFP-Sense 87 AAACATCTGGATGCGTTGGTGGCAGATGAAGATCTGAGCCGTTTTATCCTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hslU-EGFP-AntiSense 72 TGGGGCCTTTCAGCCCCATCAAACAATGATGAAAATGATTGAACGCGATTATAGTGAACCTCTTCGAGGGAC hslU Sense Primer 21 GAGATTTCCTACGACGCCAGC hslU Antisense Primer 23 GTTGTTCAGTCATAATACGCGCC hslV-EGFP-Sense 87 TGCATCTATACCAACCATTTCCACACCATCGAAGAATTAAGCTACAAAGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hslV-EGFP-AntiSense 72 CAGTTCGCTGACGATTTCGCGTGGGGTCATTTCAGACATGGGAGATCCTTATAGTGAACCTCTTCGAGGGAC hslV Sense Primer 21 TTGCTGAAAAGGCGTTGGATA hslV Antisense Primer 21 CAGCTCTTCGTTGAGCTGCAT htpG-EGFP-Sense 87 CTGGAAGATCCGAACCTGTTTATTCGTCGTATGAACCAGCTGCTGGTTTCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 186 htpG-EGFP-AntiSense 72 TGATGAAAAGAAAAATGCCGGATGACACGAAGGTCATCCGGCATTACATCATAGTGAACCTCTTCGAGGGAC htpG Sense Primer 21 AGATGAAGCGAAGTTCAGCGA htpG Antisense Primer 21 TAATGGCGAGATAATTTGCGG htpX-EGFP-Sense 87 CCGCCGCTGGATAAACGAATTGAAGCTCTGCGTACGGGTGAATACCTGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG htpX-EGFP-AntiSense 72 TAAAAAAAGCGCGTCGATCAGGACGCGCTTTTTAGTATTTACTTCATATTATAGTGAACCTCTTCGAGGGAC htpX Sense Primer 23 TCGCTCAGTGAGTTGTTCATGAC htpX Antisense Primer 21 CGTATCACTCAGCCACGATCC hupB-EGFP-AntiSense 72 CAGTAGATGCGCCCTTGAACTTCGTCACATCCCCACTGGGGACAACGCTTATAGTGAACCTCTTCGAGGGAC hupB-EGFP-Sense 87 GCTAAAGTACCGAGCTTCCGTGCAGGTAAAGCACTGAAAGACGCGGTAAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG hupB Sense Primer 22 ACCGGTAAAGAGATCACCATCG hupB Antisense Primer 25 GTCACCGAATACAAATAAAAAAGGC ihfA-EGFP-Sense 87 CCCGGGCAGAAGTTAAAAAGCCGGGTCGAAAACGCTTCGCCCAAAGACGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ihfA-EGFP-AntiSense 72 CAGTGAAAAGAAAAAAGGCCGCAGAGCGGCCTTTTTAGTTAGATCAGATTATAGTGAACCTCTTCGAGGGAC ihfA Sense Primer 22 GGATATTCCCATTACAGCACGG ihfA Antisense Prime 22 GCGTTGATTTTACGGTGACTCT IhfB-EGFP-Sense 87 CCTCACTTTAAACCTGGTAAAGAACTGCGCGATCGCGCCAATATTTACGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG IhfB-EGFP-AntiSense 72 AAAGCACCCGACAGGTGCTTTTCTCTCGTTCAAGTTTGAGTAAAAAACTTATAGTGAACCTCTTCGAGGGAC ihfB Sense Primer 22 CCGAAGACTGGCGATAAAGTAG ihfB Antisense Primer 24 AGCTTCCAGTTCCAGATTAGAGAA ileS-EGFP-Sense 87 GGCCGCTGTGTCAGCAACGTCGCCGGTGACGGTGAAAAACGTAAGTTTGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ileS-EGFP-AntiSense 72 CCACCAGCCACAGCCAGCGTAGCCCTGTTGAACAGATCGATTGACTCATCATAGTGAACCTCTTCGAGGGAC ileS Sense Primer 20 GCGTTGAGTAAAGCCGAAGG ileS Antisense Primer 22 AGCAAAGTTCTGGAGGATCAGG ilvD-EGFP-Sense 87 GCAACCAGCGCCGACAAAGGCGCGGTGCGCGATAAATCGAAACTGGGGGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ilvD-EGFP-AntiSense 72 ATTCGGCACCTTCCGGAGCACCGGACAGGGGTTGCGAGTCAGCCATTATTATAGTGAACCTCTTCGAGGGAC ilvD Sense Primer 21 TGAACGTCAGGTCTCCTTTGC ilvD Antisense Primer 21 TTTTTGTAGCGGCGTAACCTG infA-EGFP-Sense 87 GAACTGACCCCGTACGACCTGAGCAAAGGCCGCATTGTCTTCCGTAGTCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 187 infA-EGFP-AntiSense 72 CCGACCTTTTACTCGTTCTTTCTCTTCGCCCATCAGGCGGTAAAACAATCATAGTGAACCTCTTCGAGGGAC infA Sense Primer 20 CTACATCCGCATCCTGACGG infA Antisense Primer 27 GGAAGTATAAGTCCGTAACTTGTCTCG infB-EGFP-Sense 87 GGCGATGTGATCGAAGTATTCGAAATCATCGAGATCCAACGTACCATTGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG infB-EGFP-AntiSense 72 TGCCGGATGCAGCGTAAACGCCTTATCCCGCATGGAACCCTAAAAACCTTATAGTGAACCTCTTCGAGGGAC infB Sense Primer 23 TTAAGAACTACAACGACGTCCGC infB Antisense Primer 19 CCAGCGTCACATCAGGCAA ispA-EGFP-Sense 87 GATACCTCGGCACTGGAAGCGCTAGCGGACTACATCATCCAGCGTAATAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ispA-EGFP-AntiSense 72 GTATTTGGCAATATCAAAACTCATCAGGGGCCTATTAATACTTATTGTTTATAGTGAACCTCTTCGAGGGAC ispA Sense Primer 21 CCGTCAGTCGCTGAAACAACT ispA Antisense Primer 20 CTGGGTGGAGTCGACCAGTG ispB-EGFP-Sense 87 TGGCGAGAAGCACTCATCGGCCTCGCGCACATCGCTGTTCAACGCGATCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ispB-EGFP-AntiSense 72 GAACTTACTGGAACCATCCCGCGCGCTGCGCGGGATGAGGGGAGGGGATTATAGTGAACCTCTTCGAGGGAC ispB Sense Primer 19 AGCAGACAAAGCCATCGCA ispB Antisense Primer 25 CTATACTCCTGATGGCCTATTGCTC kdsA-EGFP-Sense 87 AAAGCGATTGATGATCTGGTGAAAGGTTTCGAAGAACTGGATACCAGCAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG kdsA-EGFP-AntiSense 72 AAAGTCTTAACGCAGAACGCTAATACTTTATTTTTCAAGCAAAAAAGATTATAGTGAACCTCTTCGAGGGAC kdsA Sense Primer 20 TGGAACCGTTCCTCAAGCAG kdsA Antisense Primer 19 GCCTGCGGCCTTTTTTGTA kdsB-EGFP-Sense 87 ACAGGTGTGGATACCCCTGAAGATCTTGAGCGCGTTCGCGCTGAAATGCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG kdsB-EGFP-AntiSense 72 ACGGTACGACACTCCTCCCAAAATTGGCTGAAGTGTCGTGAAGTGAAATTATAGTGAACCTCTTCGAGGGAC kdsB Sense Primer 23 ATGTTGCTGTTGCTCAGGAAGTT kdsB Antisense Primer 25 GTCACCTGAATGAGATCAATTGTTG kdtA-EGFP-Sense 87 CTACAGCGTCTGCTTCAACTGCTGGAACCTTACCTGCCACCGAAAACGCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG kdtA-EGFP-AntiSense 72 ATGGGATCGAAAGTACCCGGATAAATCGCCCGTTTTTGCATAACAACCTCATAGTGAACCTCTTCGAGGGAC kdtA Sense Primer 21 TTACTCACCGACGCCGATTAC kdtA Antisense Primer 22 CGCGTCACGATATCGATATGAC kdtB-EGFP-Sense 87 ACCCATTTCCTGCCGGAGAATGTCCATCAGGCGCTGATGGCGAAGTTAGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 188 kdtB-EGFP-AntiSense Prime 72 CCAGAAGTAATTCATGCGCGCCGGATGGCATACCATCCGGCATAAACGCTATAGTGAACCTCTTCGAGGGAC kdtB Sense Primer 22 TTCATCGTTGGTGAAAGAGGTG kdtB Antisense Primer 20 TTCATCGTTGGTGAAAGAGGTG ksgA-EGFP-Sense 87 TATTGCCAGATGGCGAACTATCTGGCGGAGAACGCGCCTTTGCAGGAGAGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ksgA-EGFP-AntiSense 72 TGTAGACGCTTTGAACCTGAATACACACTCGGGGCGAATTGATCATCGTTATAGTGAACCTCTTCGAGGGAC ksgA Sense Primer 21 GGAAAATATCTCTGTCGCGCA ksgA Antisense Primer 23 TGCGTATGGTTACGGTATAAGCA lepA-EGFP-Sense 87 CTGCCGCAGGAAGCGTTCCTCGCCATTCTGCACGTCGGCAAAGACAACAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lepA-EGFP-AntiSense 72 AATCACCAGAATCAGGGCAAACATATTCGCCATGCCAACTCCTAAGGGTTATAGTGAACCTCTTCGAGGGAC lepA Sense Primer 23 AAGGTAAGAAACGCATGAAGCAG lepA Antisense Primer 21 CATAAAATGCCCGTCACCAGT lepB-EGFP-Sense 87 GAAGGCGAATGGCCGACTGGTCTGCGCTTAAGTCGCATTGGCGGCATCCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lepB-EGFP-AntiSense 72 TAATTCACGTTGTCGCCATAACGGCGACAACGTGAACGAAGATGGCTATTATAGTGAACCTCTTCGAGGGAC lepB Sense Primer 18 CTTTGTGCCGGAAGCGAA lepB Antisense Primer 23 TTAGCCACGGGAGATTTATCTCA leuS-EGFP-Sense 87 GTACGTAAAGTGATTTACGTACCAGGTAAACTCCTCAATCTGGTCGTTGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG leuS-EGFP-AntiSense 72 CCAGAGATAACAACAATGTTGCCAGATATCGCACGCTTCCTCCCGCGCTTATAGTGAACCTCTTCGAGGGAC leuS Sense Primer 21 GCCAGGAACATCTGGTAGCAA leuS Antisense Primer 21 TCATAGTGGAAGGAACCTGCG lexA-EGFP-Sense 87 TTCACCATTGAAGGGCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lexA-EGFP-AntiSense 72 TGAAAAACAAACCGCGACGCCAGGCGGCATCGCGGTCTCAGAGATATGTTATAGTGAACCTCTTCGAGGGAC lexA Sense Primer 23 GCGAGTTTAAACCAATTGTCGTT lexA Antisense Primer 20 AATGCCATGCAGACAAGCCT lgT-EGFP-Sense 87 GTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lgT-EGFP-AntiSense 72 TTCGTCGAGCACTTTTTGCATCAGTTCTAAATACTGTTTCATGGTTCCTCATAGTGAACCTCTTCGAGGGAC lgT Sense Primer 23 AAATTCTTTCCATCCCGATGATT lgT Antisense Primer 21 CGGTACGGTCGTTTTTCTGTG ligA-EGFP-Sense 87 CTGGGCATTGAAGTCATCGACGAAGCGGAAATGCTGCGTTTGCTGGGTAGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 189 ligA-EGFP-AntiSense 72 AACGGCATTATCGTATTGGCTATTTCAATCAGCTGCTCTTTTTCCATCTCATAGTGAACCTCTTCGAGGGAC ligA Sense Primer 21 TGCAGGATCTAAACTGGCGAA ligA Antisense Primer 22 TTAAGCGACGCCCTTTGTATTT lipA-EGFP-Sense 87 GTCCGCTCTTCTTACCACGCCGATTTGCAGGCGAAAGGGATGGAAGTTAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lipA-EGFP-AntiSense 72 TCAAAAAGCGGGTTTTTTATCAGACAGATGTAAGTAATTATTACAGGATTATAGTGAACCTCTTCGAGGGAC lipA Sense Primer 19 GAAATGAAAGCCGAAGCGC lipA Antisense Primer 19 AAGAGTGACGTGGCGAGCA lipB-EGFP-Sense 87 GAAAATATTTTAGCGCTACTAAACAATCCGGACTTCGAATATATTACCGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lipB-EGFP-AntiSense 72 ATATTATGAGTAATGACCCAGTGTAAATTGGGCCATTGATGTATGGAATTATAGTGAACCTCTTCGAGGGAC lipB Sense Primer 20 GAAACCCGAAGCGACGACTA lipB Antisense Primer 21 ACGCGAAGGAGATGTAAAGCA lolA-EGFP-Sense 87 AAATTTACCTTCACCCCGCCGCAAGGCGTCACGGTAGATGATCAACGTAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lolA-EGFP-AntiSense 72 AAAGTATTATCCGAAAAATCGAGCGACAGATTGCTCACTCAGGTGCCTCTATAGTGAACCTCTTCGAGGGAC lolA Sense Primer 24 CAGCAGTTATCAACTGAAATCCCA lolA Antisense Primer 25 CTGGCCGATATACTGTGCTAAATTT loN-EGFP-Sense 87 ACTCTGGCGCTGCAAAATGAACCGTCTGGTATGCAGGTTGTGACTGCAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG loN-EGFP-AntiSense 72 AATTAGCCTGCCAGCCCTGTTTTTATTAGTGCATTTTGCGCGAGGTCACTATAGTGAACCTCTTCGAGGGAC loN Sense Primer 21 TGTGAAGCGCATTGAGGAAGT loN Antisense Primer 22 CCGCCATCTAACTTAGCGAGAC lpD-EGFP-Sense 87 GTGTTCGAAGGTAGCATTACCGACCTGCCGAACCCGAAAGCGAAGAAGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lpD-EGFP-AntiSense 72 CGTGGTTAGCCGCTTTTTTAATTGCCGGATGTTCCGGCAAACGAAAAATTATAGTGAACCTCTTCGAGGGAC lpD Sense Primer 19 TCTGCACGAGTCTGTGGGC lpD Antisense Primer 23 ACTGGAAAGGTAAATTGCAGACG lpxA-EGFP-Sense 87 GTGAAAGCCTTTACCGATTTCTTTGCACGCTCAACGCGCGGTCTGATTCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lpxA-EGFP-AntiSense 72 AGGTTTCTCCGGCGACCAGGGCAATCGTTAATGGACGCTGTTCAGTCATTATAGTGAACCTCTTCGAGGGAC lpxA Sense Primer 21 GTGAAACCGGAAATTGCTGAA lpxA Antisense Primer 21 GGCACATGTTCTTTCAGAGCG lpxB-EGFP-Sense 87 CGCTGCAATGCCGATGAGCAGGCGGCACAAGCCGTTCTGGAGTTAGCACAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 190 lpxB-EGFP-AntiSense 72 CTTCATCCACACCCGCAACCAGCTGCGTGTGCGGATAAACAAATTCGATCATAGTGAACCTCTTCGAGGGAC lpxB Sense Primer 19 CACGCGATGCACGATACCT lpxB Antisense Primer 18 AAGGATCACCGCAGCGGT lpxC-EGFP-Sense 87 GACGACGCAGAACTGCCGTTGGCCTTCAAAGCGCCTTCAGCTGTACTGGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lpxC-EGFP-AntiSense 72 AGAGTGCCAGATTTGCCAGTCGAATTTTATACGACAGTATAAATGTCGTTATAGTGAACCTCTTCGAGGGAC lpxC Sense Primer 20 AAACTGCTGCAGGCTGTCCT lpxC Antisense Primer 22 AAAAAACGACTGGTTCACCTGG lpxD-EGFP-Sense 87 GACATGAGCAAGCGTCTGAAATCGCTTGAGCGCAAGGTTAATCAACAAGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lpxD-EGFP-AntiSense 72 CGTAAGAATGAGACAGGCCGTAAAGTTTGGCGAACAAAAGATGGAACGTTATAGTGAACCTCTTCGAGGGAC lpxD Sense Primer 19 CCAACAAAGTCTGGCGCAA lpxD Antisense Primer 23 AACGACAATAATAACACGGCCTG lpxK-EGFP-Sense 87 GATGAACCAGCGAAACTGCTTACGCAACTAACCTTGCTGGCTTCTGGCAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lpxK-EGFP-AntiSense 72 CGGCAGCGACATTCATGACTCCATCAATCGAACGCTGCCGCGGCGTAACTATAGTGAACCTCTTCGAGGGAC lpxK Sense Primer 22 TATTTGCCTGTAGACGCACAGC lpxK Antisense Primer 21 AAGATTACGCGCATCAGCAAG lspA-EGFP-Sense 87 GCGGCACTGATTGTGCTGGAAGGTTTTTTGCCTTCTAGAGCGAAAAAACAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lspA-EGFP-AntiSense 72 ATCTGTAGGCCGGATAAGATGCGTCAGCATCGCATCCGGCAGGGTTTATTATAGTGAACCTCTTCGAGGGAC lspA Sense Primer 21 GCCGATACTGCCATCTGTGTC lspA Antisense Primer 20 GCCTTATCCGGCCTACGATT lytB-EGFP-Sense 87 ATTGTTTTCGAAGTGCCGAAAGAGCTGCGTGTCGATATTCGTGAAGTCGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG lytB-EGFP-AntiSense 72 TGCCGGTAACAAGACCGGCATTTTCGCATAACTTAGGCTGCTAATGACTTATAGTGAACCTCTTCGAGGGAC lytB Sense Primer 24 GATATTCTGGTGCAGAATGTGGTG lytB Antisense Primer 24 TAGGTAAACGCATGTTTTCTCCAT mazG-EGFP-Sense 87 GAAACAATGGAAGAAGTCTGGCAACAGGTAAAACGGCAGGAAATTGATCTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mazG-EGFP-AntiSense 72 CTTGTCATTTAAAAAATGACACAAATGGCGCTTGACCGCGTAATTCCCTTATAGTGAACCTCTTCGAGGGAC mazG Sense Primer 21 CGAAGTGGAGCGTATTGTTGC mazG Antisense Primer 24 GGTAAATGTTTTTGACGCAAATCA menG-EGFP-Sense 87 TATGCCGACAATACCGGGATTATTCTTTCAGAAGATCCGCTGGATATTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 191 menG-EGFP-AntiSense 72 AGGCCAACAGGTAACGCAGAAAAAAGGCACCTTGCGGTGCCTTTCTTATCATAGTGAACCTCTTCGAGGGAC menG Sense Primer 18 AAAGCGATGTCCGCGTCA menG Antisense Primer 22 GCTTCATCTTCCGCGTAATTTC metB-EGFP-Sense 87 GAAGATTTAATTGCCGACCTGGAAAATGGCTTCCGGGCTGCAAACAAGGGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG metB-EGFP-AntiSense 72 TATGCAGCTGACGACCTTTCGCCCCTGCCTGCGCAATCACACTCATTTTTATAGTGAACCTCTTCGAGGGAC metB Sense Primer 23 CGTATCTCCACCGGTATTGAAGA metB Antisense Primer 22 TCACATCAGCCAGACTACTGCC metF-EGFP-Sense 87 GCTGAAATGAGTTACGCGATTTGCCATACGCTGGGGGTTCGACCTGGTTTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG metF-EGFP-AntiSense 72 TGTTTAAAATTTGTGATCACTGTGTGATTTTCACAAAAGCCACACTATTTATAGTGAACCTCTTCGAGGGAC metF Sense Primer 23 TTAAGCCGTGAAGGAGTGAAAGA metFAntisense Primer 20 CCTCATTTCGAGGCAGCATT metG-EGFP-Sense 87 ATTTTCCTGCTAAGCCCGGATGCCGGTGCTAAACCGGGTCATCAGGTGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG metG-EGFP-AntiSense 72 GGAATTTATAAAGAAAAGGCGCTGTCGATGCAGCGCCTTGAAGGGGGATTATAGTGAACCTCTTCGAGGGAC metG Sense Primer 21 TCGGTATCTCTGAAGGCATGG metG Antisense Primer 23 CAGTTGAATGCAGATGCTACCAG mfD-EGFP-Sense 87 ATCGAATGGGTACGCCAGTTTATGCGTGAACTGGAAGAGAACGCGATCGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mfD-EGFP-AntiSense 72 ATTTGTAAATGTTGCAGATGGGGGCGCAGAAACGCCCCCGATTTACCATTATAGTGAACCTCTTCGAGGGAC mfD Sense Primer 21 TTCAGGATTTGAGTGAGCGGA mfD Antisense Primer 24 TAAGCCTGAATTATGGATGGTGAC miaA-EGFP-Sense 87 CCAGAACAGGCGCGTGACGAAGTATTACAGGTTGTTGGTGCTATCGCAGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG miaA-EGFP-AntiSense 72 CTTTCGATTCTGAAAAAATTGCGCACGATACGTCTCAATTGTACACATTCATAGTGAACCTCTTCGAGGGAC miaA Sense Primer 21 AGATAACCTGGCTGCGTGGTT miaA Antisense Primer 23 CTTGTAAAGATTGCCCCTTAGCC minD-EGFP-Sense 87 CGCTTCATTGAAGAAGAGAAGAAAGGCTTCCTCAAACGCTTGTTCGGAGGACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG minD-EGFP-AntiSense 72 GGCTGTGTTTTTCTTCCGCGAGAGAAAGAAATCGAGTAATGCCATAACTTATAGTGAACCTCTTCGAGGGAC minD Sense Primer 23 CGTAGAACGTCTGTTGGGAGAAG minD Antisense Primer 22 CAACAATAATCTGCAGCCGTTC mpL-EGFP-Sense 87 ATCCATCAGAAACTGCTGGATGGGCTGGCGAAGAAGGCGGAAGCCGCGCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 192 mpL-EGFP-AntiSense 72 GTGAACGCCTTATCCGGCCTACGCATACTATATCTGGCTAAGGCCGAATTATAGTGAACCTCTTCGAGGGAC mpL Sense Primer 19 TCTGGTGATGAGCAACGGC mpL Antisense Primer 21 GCCTGATAAGACGCATCAAGC mraY-EGFP-Sense 87 ATTATTTCGCTGATGCTGGTTCTGATTGGTCTGGCAACGCTGAAGGTACGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mraY-EGFP-AntiSense 72 GGCCCAGGCCGATAATGACGACATTTTTACCCTGATAATCAGCCATGATTATAGTGAACCTCTTCGAGGGAC mraY Sense Primer 23 ACCACTATGAACTGAAAGGCTGG mraY Antisense Primer 21 AAAAAGTCCACGCAGGAAAGC mrcA-EGFP-Sense 87 CACGAGGTGGGAACGACCATTATCGATAATGGCGAGGCACAGGAATTGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mrcA-EGFP-AntiSense 72 AAGTGCACTTTGTCAGCAAACTGAAAAGGCGCCGAAGCGCCTTTTTAATCATAGTGAACCTCTTCGAGGGAC mrcA Sense Primer 20 GACACAACAGGCAGTGCACG mrcA Antisense Primer 19 AGCCGGATAACGCGTTCAC mrcB-EGFP-Sense 87 AAAGACAGCGACGGTGTAGCCGGTTGGATCAAGGATATGTTTGGTAGTAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mrcB-EGFP-AntiSense 72 TTATTTTACCGGATGGCAACTCGCCATCCGGTATTTCACGCTTAGATGTTATAGTGAACCTCTTCGAGGGAC mrcB Sense Primer 21 AACAGCAACCTGCTCAGCAAG mrcB Antisense Primer 25 CGTTCCTGAAGGGTTAATAAACAAC mrdA-EGFP-Sense 87 AACACCGATCTGCCTGCGGAAAATCCAGCGGTTGCCGCAGCGGAGGACCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mrdA-EGFP-AntiSense 72 GATGGACTTTATCCCAGAATGTTTTTTTATTCGGATTATCCGTCATGATTATAGTGAACCTCTTCGAGGGAC mrdA Sense Primer 23 CAGATCCTCGACCACATTATGCT mrdA Antisense Primer 23 CAGTAAGATCAGCAGCATTGTGG mrdB-EGFP-Sense 87 GGGATTGTAATGTCAATCCACACCCACAGGAAAATGTTGTCGAAAAGCGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mrdB-EGFP-AntiSense 72 CCTGCCGCGATGCAGATCCCGAGCCACTGCTTACGCATTGCGCACCTCTTATAGTGAACCTCTTCGAGGGAC mrdB Sense Primer 21 CTAATTGTGCTGATGGCTGGG mrdB Antisense Primer 22 GACCATCATCGCTTGTACATGC mreB-EGFP-Sense 87 AAAGCGCTGGAAATGATCGACATGCACGGCGGCGACCTGTTCAGCGAAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mreB-EGFP-AntiSense 72 GTATCAGACCAGGCAGGGTAAACAGACACTTCCCCTGCCTGCATCCGATTATAGTGAACCTCTTCGAGGGAC mreB Sense Primer 22 TTCCAGTCGTTGTTGCTGAAGA mreB Antisense Primer 25 AAAATTGGCTTCATAAGTTATGCGT mreC-EGFP-Sense 87 ACGCCGCCGCAAAGTGGTGCTCAACCGCCTGCGCGTGCGCCGGGAGGGCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 193 mreC-EGFP-AntiSense 72 GGAAAGAGAGCCAGATTACCCAGCGTCCCTGGCTACGATAGCTCGCCACTATAGTGAACCTCTTCGAGGGAC mreC Sense Primer 21 GCTGCTAATCGCTCTCCACAA mreC Antisense Primer 22 GCCGGAAAACAATCAGGTTATC mreD-EGFP-Sense 87 CCGTGGATTTTCTTGCTGATGCGCAAAGTCCGTCAGCAGTTTGCAGTGCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mreD-EGFP-AntiSense 72 GACGACGCGGAGAACCGGAAGCTAAATACAGAGAAGTCATAGAAACCTTTATAGTGAACCTCTTCGAGGGAC mreD Sense Primer 23 ACCGGAAGTGTTCTGGAGTAGTG mreD Antisense Primer 23 CCCGTAACAATACGTTCAAAGGT mrP-EGFP-Sense 87 CTCTACTGGCAGGGTGAAGTCATTCCAGGCGAGATTTCCTTCCGCGCGGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mrP-EGFP-AntiSense 72 GTAACATCTCTGCGGTGTATGAAGAATAACCAGAATGGTTAATAGGCGTTATAGTGAACCTCTTCGAGGGAC mrP Sense Primer 23 GGAGAGCGAATTTACCGCTATCT mrP Antisense Primer 21 CGGCAAAAACTAATACACCGC msbA-EGFP-Sense 87 CTTGAGCACCGCGGCGTTTACGCGCAACTTCACAAAATGCAGTTTGGCCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG msbA-EGFP-AntiSense 72 GCAGCAATAGCCGCCACAAAGGGGATTCACCAGACCAGATTTTTTCGATCATAGTGAACCTCTTCGAGGGAC msbA Sense Primer 20 AATCGTGGTCGTCGAGGATG msbA Antisense Primer 21 CACTCACCAGGCCATACAACC murA-EGFP-Sense 87 GAAGACAAACTGCGCGCTTTAGGTGCAAATATTGAGCGTGTGAAAGGCGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG murA-EGFP-AntiSense 72 AGTGGTCTGGCGGTAGCCCCGCGAACGGGGCTGCCAGCTCTCAGACGATTATAGTGAACCTCTTCGAGGGAC murA Sense Primer 25 GTGGTTGATCGTATTTATCACATCG murA Antisense Primer 25 GCAGTTGATGCGTAGCTACACTAAA murB-EGFP-Sense 87 GTCCGCTTTATTGGTGCATCAGGTGAAGTGAGCGCAGTGGAGACAATTTCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG murB-EGFP-AntiSense 72 CACCGTTCGCTAACAGGGCAATCAATTTCAGTGGCACGGTGTTATCCTTCATAGTGAACCTCTTCGAGGGAC murB Sense Primer 20 CAGCTGGCGCATCATGTAAG murB Antisense Primer 19 ACCCAACTGCTCGCCAGAG murC-EGFP-Sense 87 TTAGCTGAAATCAAACTGAAGCCGCAAACTCCGGAGGAAGAACAACATGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG murC-EGFP-AntiSense 72 GAAACTTCCCGCTCAGCGGAGGTCCCACCCAACAGGACCGCGATTTTATCATAGTGAACCTCTTCGAGGGAC murC Sense Primer 24 GTAATATTGGAAAAATTGCCCGTT murC Antisense Primer 18 CGCGTCAATACCGCCTTC murD-EGFP-Sense 87 AACTTTGAACAACGAGGCAATGAGTTTGCCCGTCTGGCGAAGGAGTTAGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 194 murD-EGFP-AntiSense 72 TGAATCCTGGCAGGCGCGGCATTTTCAGGCGAGGGAGAGATAAACGCATCATAGTGAACCTCTTCGAGGGAC murD Sense Primer 19 TGGTTCTGCTCTCCCCAGC murD Antisense Primer 18 TAGCGCCGTGGAGATCCA murE-EGFP-Sense 87 CTGGACTACTCCGATCGCGTCACGGTGGCGCGTCTGCTGGGGGTGATTGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG murE-EGFP-AntiSense 72 GCAGTTCACCGTTGAGAATGTCGGTAAGTTGGCTAAGGGTTACGCTAATCATAGTGAACCTCTTCGAGGGAC murE Sense Primer 24 CCATGAAGATTACCAGATTGTTGG murE Antisense Primer 24 TACAGCATCAAGGGTGATATCTGC murF-EGFP-Sense 87 AGTGCCGCCATGGAAGAGGTAGTACGCGCTTTACAGGAGAATGGGACATGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG murF-EGFP-AntiSense 72 AGACGTTAAAGCCGGAATAATATTTGACCAAATGTTCGGCCAGCCAAACTATAGTGAACCTCTTCGAGGGAC murF Sense Primer 27 AATTACGATTTTAGTTAAGGGTTCACG murF Antisense Primer 22 CCCATCCACAATGAGATGAACA murG-EGFP-Sense 87 CCGGATGCCACCGAGCGAGTGGCAAATGAAGTGAGCCGGGTTGCCCGGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG murG-EGFP-AntiSense 72 ACGCCATTAACTTCTTAAATTCATACGATGCAAAAGGCATCGCTACAATTATAGTGAACCTCTTCGAGGGAC murG Sense Primer 23 ACCTTATTAACCATGGCAGAACG murG Antisense Primer 25 CAGTTTTGCCAATTGTTGTGTATTC murI-EGFP-Sense 87 GTTTTACAGCGTTACGGCTTCGAAACGCTCGAAAAACTGGCAGTTTTAGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG murI-EGFP-AntiSense 72 CAAGAGGAAATTTAAAATAATTTTCTGACCGCGCAACATTCAACCAAATCATAGTGAACCTCTTCGAGGGAC murI Sense Primer 24 AATATTGCCTTTTGTATGGCAATG murI Antisense Primer 23 GGCGCATTATAGGGAGTTATTCC mutL-EGFP-Sense 87 CTGTTACAATCTGTTGATTTACATCCGGCGATAAAAGCCCTGAAAGATGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mutL-EGFP-AntiSense 72 GTCGGCCCCATCAAAAAAATCGCCTTAGGCAGGCTCGCCTTACTGATATCATAGTGAACCTCTTCGAGGGAC mutL Sense Primer 21 AACGGTTATGTCCGCAACTTG mutL Antisense Primer 21 CAATGGCTAACGCCGTTTTAC mutM-EGFP-Sense 87 GCGACTAAACATGCGCAGCGGGCAACGTTTTATTGTCGGCAGTGCCAGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mutM-EGFP-AntiSense 72 GTTAGCGTAGCGTTTATGCCGGATGGTATGCCATCCGGCGCGCATGAATTATAGTGAACCTCTTCGAGGGAC mutM Sense Primer 23 AAAGTGATGGTAAACCGGGCTAT mutM Antisense Primer 20 AATGTCCATCAGGCGCTGAT mutS-EGFP-Sense 87 CTCACCCCGCGTCAGGCGCTGGAGTGGATTTATCGCTTGAAGAGCCTGGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 195 mutS-EGFP-AntiSense 72 CAGTTGTCGTTAATATTCCCGATAGCAAAAGACTATCGGGAATTGTTATTATAGTGAACCTCTTCGAGGGAC mutS Sense Primer 23 CGCAAATGTCTTTGCTGTCAGTA mutS Antisense Primer 25 GGCATGGTTTTACCCTGTAAAATAA mutY-EGFP-Sense 87 GCGGCTCCCGTGGAGCGTTTGTTACAGCAGTTACGCACTGGCGCGCCGGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mutY-EGFP-AntiSense 72 ACAAAAAATCGTTCTGCTCATAAATCATCCTCTTTATCGACTCACGCGCTATAGTGAACCTCTTCGAGGGAC mutY Sense Primer 20 GCTGCATGGATGAAGGCAAT mutY Antisense Primer 20 TTCTGCTTCACGTTGCAGGA mvN-EGFP-Sense 87 GCACTGGCGGTACTGGGCTTCAAAGTTAAAGAATTTGCCCGCCGGACGGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG mviN-EGFP-AntiSense 72 TCGATCTGAAGATTATCTCCGGCCTGCACTGCAGGCCGGAATGCATTGTTATAGTGAACCTCTTCGAGGGAC mviN Sense Primer 22 ATATCATGCCGGAGTGGTCATT mviN Antisense Primer 20 CTATATGGGGCGAACGGTCA ndK-EGFP-Sense 87 CGCGAAATCGCTTATTTCTTTGGCGAAGGCGAAGTGTGCCCGCGCACCCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ndK-EGFP-AntiSense 72 AATTCTGGCGCACGGATGCCACGTTTGCACGCGGCATTTACGAAATTATTATAGTGAACCTCTTCGAGGGAC ndK Sense Primer 20 CTGATTCCGTCGAATCTGCC ndK Antisense Primer 22 TGGAGGGTTGAAAAAAGAAACG ntH-EGFP-Sense 87 GGCTCTTGTATTATTGAAGATCTTTGTGAATACAAAGAGAAAGTTGACATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ntH-EGFP-AntiSense 72 AGGATAAAGAAAGGTTATCAATGGGGTAATCGGTGTTACCCCTTTTCTTCATAGTGAACCTCTTCGAGGGAC ntH Sense Primer 20 CACGGGCGTTATACCTGCAT ntH Antisense Primer 25 TACCTGATGATAACGCTAAGCAATG nusB-EGFP-Sense 87 GTCAACGGCGTACTCGATAAAGCAGCACCTGTGATTCGCCCTAACAAAAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG nusB-EGFP-AntiSense 72 AGGCCGGCGATCATGGAACGGTCTTCCGTGAATCTACCGGCCTGGATATCATAGTGAACCTCTTCGAGGGAC nusB Sense Primer 21 TTAACGAAGCGATCGAACTGG nusB Antisense Primer 22 ACATGCCATACGTTATGCCTCA nusG-EGFP-Sense 87 TTCGGTCGTGCGACCCCGGTAGAGCTGGACTTCAGCCAGGTTGAAAAAGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG nusG-EGFP-AntiSense 72 TCTCACGCCTTGTGCAACGATTAAATCGCCGCTTTTTTGATCGCTGGGTTATAGTGAACCTCTTCGAGGGAC nusG Sense Primer 25 GGATTACGAGAAATCTCGTCTGAAA nusG Antisense Primer 21 AACAAAAGGCGCGAAATTGTA ogT-EGFP-Sense 87 GGAGTTCAGCGAAAAGAGTGGTTATTGCGCCATGAAGGTTATCTTTTGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 196 ogT-EGFP-AntiSense 72 GTAACTTTACATAAAAGTGTGAACAAGCTGGCACAAATTGTTTAATGTTTATAGTGAACCTCTTCGAGGGAC ogT Sense Primer 20 ACCATGACCGGATATGCAGG ogT Antisense Primer 26 ACAGCAGAGAATTCCGATATTAGATG orN-EGFP-Sense 87 CGTGAATCGGTGGCGGAGCTGGCTTACTACCGCGAGCATTTTATCAAGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG orN-EGFP-AntiSense 72 TCACGCCGCCGCAAGTGGATTCCGGCGCTTACGTGACCAGGAAAAATTTTATAGTGAACCTCTTCGAGGGAC orN Sense Primer 19 TTTACCAAGCAGGGGACGC orN Antisense Primer 23 AGCAAGACGTTCAATTTAGCGTC oxyR-EGFP-Sense 87 ATCCGCGCAAGAATGGATGGCCATTTCGATAAAGTTTTAAAACAGGCGGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG oxyR-EGFP-AntiSense 72 AACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAACGGTTTATAGTGAACCTCTTCGAGGGAC oxyR Sense Primer 19 CGCTATGAGCAGCTGGCAG oxyR Antisense Primer 21 GTGGCGGCAACACTATTGAGT pabA-EGFP-Sense 87 AGTATTCTTAGCGAACAAGGACATCAACTGCTGGCTAATTTCCTGCATCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pabA-EGFP-AntiSense 72 TTATAACCACAAAATATGCATAAAAAATCACTAAATGGCAATCAGAAATCATAGTGAACCTCTTCGAGGGAC pabA Sense Primer 21 TGGAAGGTGTGCAGTTCCATC pabA Antisense Primer 24 ATGATCACCCTGTTACGCATAAAC paL-EGFP-Sense 87 GGTCATGACGAAGCGGCATACTCCAAAAACCGTCGTGCGGTACTGGTTTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG paL-EGFP-AntiSense 72 CGACAGACTCAATAGTTGATGTCTGAAGTTACTGCTCATGCAATTCTCTTATAGTGAACCTCTTCGAGGGAC paL Sense Primer 25 ATCTCCATCGTTTCTTACGGTAAAG paL Antisense Primer 20 TGATTGGTGCCTGAGCAAAA panB-EGFP-Sense 87 ATGGCTGAAGTGGAGTCCGGCGTTTATCCGGGCGAAGAACACAGTTTCCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG panB-EGFP-AntiSense 72 GCTGACGCAGCAGCGGCAGGGTTTCGATAATTAACACAACGTGACTCCTTATAGTGAACCTCTTCGAGGGAC panB Sense Primer 23 CGGTCACATTCCTAAATTCGCTA panB Antisense Primer 20 CCATCGTGCAGGTTACCCAT panC-EGFP-Sense 87 GCCTGGCTTGGCGATGCTCGCCTGATCGACAACAAAATGGTCGAGCTGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG panC-EGFP-AntiSense 72 TGGGGTAAATTAATAACTGGCGCCATCAGCCGTAGCGCCAGTTAAGTATTATAGTGAACCTCTTCGAGGGAC panC Sense Primer 23 CTGGAAGTTTCTGAAACCAGCAA panC Antisense Primer 21 GAAGACGTTGCAGAGATGGCA parC-EGFP-Sense 87 CGTGTTGAGATCGACTCTCCTCGCCGTGCCAGCAGCGGTGATAGCGAAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 197 parC-EGFP-AntiSense 72 ATCCGGCGTTCCTTGCAAGCGGGAGGAAACAGCGCCCTCCCCGGCATATTATAGTGAACCTCTTCGAGGGAC parC Sense Primer 18 GCGGTTTGCAGCGTATCG parC Antisense Primer 21 CCGGATGACGACTTAACGTTT parE-EGFP-Sense 87 GATCGCCGCAACTGGTTGCAAGAGAAAGGCGACATGGCGGAGATTGAGGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG parE-EGFP-AntiSense 72 ATCCTGCCTTGTTTGCCCGGCCATCCTGACCGGGCAATGTTCTTTCCTTTATAGTGAACCTCTTCGAGGGAC parE Sense Primer 20 GATATGCTGCTGGCGAAGAA parE Antisense Primer 21 GTATTCTGCAGCCAGGGATTT pcnB-EGFP-Sense 87 CGTCGTACTCGTCGTCCACGCAAACGCGCACCACGTCGTGAGGGTACCGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pcnB-EGFP-AntiSense 72 GCTCCAGCGGAGAGGCCAGATTGCTGCCTATGGCAATATACGCCACTGTCATAGTGAACCTCTTCGAGGGAC pcnB Sense Primer 20 AAGGGATGCTCAACGAGCTG pcnB Antisense Primer 21 TAATGCTTTCAGGGCAGCATT pdxA-EGFP-Sense 87 AGTTTTATTACGGCGCTTAATCTCGCCATCAAAATGATTGTTAACACCCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pdxA-EGFP-AntiSense 72 TTTGCCCGAAGCGTTTACGGGCTAAGTGGCCCTGGTGGACTCGATTATTCATAGTGAACCTCTTCGAGGGAC pdxA Sense Primer 19 GCCTGCCCTTTATTCGCAC pdxA Antisense Primer 22 TGTCGATCACGAACTGATCGTT pdxH-EGFP-Sense 87 TTTTTGTACCAGCGTGAAAATGATGCGTGGAAGATTGATCGTCTTGCACCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pdxH-EGFP-AntiSense 72 TGCCAGAATCAGGAGTACCAGCGATTAAAGCAAGATTTTTGCATCTTTTCATAGTGAACCTCTTCGAGGGAC pdxH Sense Primer 21 ATTGAGTTCTGGCAGGGTGGT pdxH Antisense Primer 23 GCCAGATGCGAAAGAGACATAGA pepA-EGFP-Sense 87 GCGTTGCTGGCACAGTTCCTGTTAAACCGCGCTGGGTTTAACGGCGAAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pepA-EGFP-AntiSense 72 ATAAGGCGTTCACGCCGCATCCGGCAATAACAGCCTTGCCTGACGCAATTATAGTGAACCTCTTCGAGGGAC pepA Sense Primer 21 CTGGCGTTCTGGTAAAGCAAA pepA Antisense Primer 24 CCTACGAGTTCAGTGCTGTGTAGG pepN-EGFP-Sense 87 CTGGAAAATCTCTCTGGCGATCTGTACGAGAAGATAACTAAAGCACTGGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pepN-EGFP-AntiSense 72 GGGTAAATTCCCCGAATGGCGGCGCTATTGCCGCCATTCGGTTATTTATCATAGTGAACCTCTTCGAGGGAC pepN Sense Primer 21 CCTGAAACGTTACGATGCCAA pepN Antisense Primer 24 TGAATCTGAAACTCGCCTGAGAA pgI-EGFP-Sense 87 CACGATAGCTCGACCAATGGTCTGATTAACCGCTATAAAGCGTGGCGCGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 198 pgI-EGFP-AntiSense 72 TGCCGGATGCGGCGTGAACGCCTTATCCGGCCTACATATCGACGATGATTATAGTGAACCTCTTCGAGGGAC pgI Sense Primer 21 GGAACTGGGTAAACAGCTGGC pgI Antisense Primer 22 GCATCGACCTGTAGGCCTGATA pgK-EGFP-Sense 87 GAAGGTAAAGTACTGCCTGCAGTAGCGATGCTCGAAGAGCGCGCTAAGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pgK-EGFP-AntiSense 72 TCTAAAAGCGCGCTGAAACAAGGGCAGGTTTCCCTGCCCTGTGATTTTTTATAGTGAACCTCTTCGAGGGAC pgK Sense Primer 21 CGGCATTGCTGACAAAATCTC pgK Antisense Primer 20 TTGTCGCCTTCCTGCAACTC pgsA-EGFP-Sense 87 TGGTCAATGTTGCAATATTTGAGCGCTGCGCGTGCAGATTTGCTTGATCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pgsA EGFP-Antisense 72 ATATTTTTCACCACTTTTGATCGTTTGCTGAAAATTACGCCGAAACGATCATAGTGAACCTCTTCGAGGGAC pgsA Sense Primer 23 GTCCGAACATTTGGGTTGAGTAC pgsA Antisense Primer 21 TTCTACTTACCTGGCGCGATG pheS-EGFP-Sense 87 GACCTGCGTTCATTCTTCGAAAACGATCTGCGTTTCCTCAAACAGTTTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pheS-EGFP-AntiSense 72 CCCATTCGCGTAACCACAGTTCACTGAATTTCATAATCTATTCCTGCCTTATAGTGAACCTCTTCGAGGGAC pheS Sense Primer 21 GGAGCGTCTGACTATGTTGCG pheS Antisense Primer 20 CCAGCGCATCGCTATCAATC pheT-EGFP-Sense 87 GCCAAATGTGTAGAGGCATTAAAAGAGCGATTCCAGGCATCATTGAGGGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pheT-EGFP-AntiSense 72 TTATCAAACAGATATTCTGACATTTCAGCTTTTGTAAGCGCCATAGGTTCATAGTGAACCTCTTCGAGGGAC pheT Sense Primer 24 CCGTACACTCGAAGAAGAGGAGAT pheT Antisense Primer 24 TCTCTTCGAAAAACAGTTCAACCA phoB-EGFP-Sense 87 CGCATGGTGCAGACCGTGCGCGGTACAGGATATCGTTTTTCAACCCGCTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG phoB-EGFP-AntiSense 72 TCCAGTTAAGAAATCATAAGCCCTGCTCTGCGTCCGATGAGCAAGGCGTTATAGTGAACCTCTTCGAGGGAC phoB Sense Primer 20 ACGGTCGATGTCCACATTCG phoB Antisense Primer 21 AGCCGTTCCAGCACGTAAGAT plsB-EGFP-Sense 87 ATTACATCAGACGTGCGTTTGACGATTGAGAGTGCGACGCAGGGCGAAGGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG plsB-EGFP-AntiSense 72 TGGAAGGCCGGATAAGGCGTTTTCGCCGCATCCGGCAATTCTCTCTGATTATAGTGAACCTCTTCGAGGGAC plsB Sense Primer 24 ACGATGAAGGTTTATCAGTTGCTG plsB Antisense Primer 23 CATCGCAATTTATTGAATTTGCA pmbA-EGFP-Sense 87 AATATACAGTGTGGTTCTGTGCTGTTGCCGGAGATGAAAATCGCCGGACAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 199 pmbA-EGFP-AntiSense 72 TTTTGTGTAATTTTTTAGTTTATAGCGCGGCAGGTCGCGCCAGTTTTTTTATAGTGAACCTCTTCGAGGGAC pmbA Sense Primer 23 ACCGTCGGTAACGATATTGAAAC pmbA Antisense Primer21 21 GGTCACGTACTGCGAGTACGC pnP-EGFP-Sense 87 CAGTCTCAACCTGCTGCAGCACCGGAAGCTCCGGCTGCTGAACAGGGCGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pnP-EGFP-AntiSense 72 CCTGCCCGGTTAAAAGCCCCCCGCCGCAGCGGAGGGCAAATGGCAACCTTATAGTGAACCTCTTCGAGGGAC pnP Sense Primer 25 GTATCCGTCTGAGCATTAAAGAAGC pnP Antisense Primer 19 GCTTCATTTCCCACTCCCG pntB-EGFP-Sense 87 ATGCTGTTTGGTGACGCCAAAGCCAGCGTGGATGCAATCCTGAAAGCTCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pntB-EGFP-AntiSense 72 TTAAGCGATCTCAATAAAGAGTGACGGCCTCAGCAGAGGCCGTCAGGGTTATAGTGAACCTCTTCGAGGGAC pntB Sense Primer 20 GTGCAAAACCCGCTGTTCTT pntB Antisense Primer 19 GAGGTCAAAGCATCGCCGT polA-EGFP-Sense 87 GTGCCGTTGCTGGTGGAAGTGGGGAGTGGCGAAAACTGGGATCAGGCGCACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG polA-EGFP-AntiSense 72 GACAGCTTATGTTGCTTACTTACGAAAAAAGGCATGTTCAGGCGAATCTTATAGTGAACCTCTTCGAGGGAC polA Sense Primer 21 AAGATGATGTTGATGCCGTCG polA Antisense Primer 26 GCATAGGAATTTCTAATAGCCATCAC ppA-EGFP-Sense 87 GAAGCCGCTAAAGCTGAAATCGTTGCCTCCTTCGAGCGCGCAAAGAATAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ppA-EGFP-AntiSense 72 CCCTTACTAACCGAAGCCCGGCGTTCAGGGTTATTACGCCAGAAGAACTTATAGTGAACCTCTTCGAGGGAC ppA Sense Primer 21 GGCAAGTGGGTGAAAGTTGAA ppA Antisense Primer 22 TGTTTATTTATCGCGGGCATAA ppdD-EGFP-Sense 87 AGCGCATTGCAGCAAGCCTGCGAAGATGTCTTCCGCTTTGATGACGCCAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ppdD-EGFP-AntiSense 72 ATAACGCAGACACAGGGCAGTGAGCTGTGGAATATTCATTGCCGCTCCTTATAGTGAACCTCTTCGAGGGAC ppdD Sense Primer 20 ACGCGCAACTGCAATATTCA ppdD Antisense Primer 20 CCGCAACATGAACCACCTCT prfA-EGFP-Sense 87 ATTATCCAGGAACATCAGGCCGACCAACTGGCGGCGTTGTCCGAGCAGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG prfA-EGFP-AntiSense 72 TCGCCTGAAGTTGGCTTATTGCTTCACGTAACCAGTGTTGATATTCCATTATAGTGAACCTCTTCGAGGGAC prfA Sense Primer 24 GAAGTGATGGAAGGTAAGCTGGAT prfA Antisense Primer 21 CCAGCAGGATTTCAGCATCAC prfC-EGFP-Sense 87 CTGGCACAGGAACGTTATCCGGACGTTCAGTTCCACCAGACCCGCGAGCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 200 prfC-EGFP-AntiSense 72 AAGGGAAATTGACAGGGCGCAGCGGCTACCGCGCCCTGGAGGCAAGAATTATAGTGAACCTCTTCGAGGGAC prfC Sense Primer 20 CATCGCTACCAGCATGGTCA prfC Antisense Primer 22 AAGAACATTCCGTAAGCGGCTA priA-EGFP-Sense 87 CCGGATTCCCGTAAGGTGAAATGGGTGCTGGATGTTGATCCGATTGAGGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG priA-EGFP-AntiSense 72 AGTGTGATGAATATTGAATTTTTCGATCCGCCTCGCATCGTGAGCGGTTTATAGTGAACCTCTTCGAGGGAC priA Sense Primer 20 TACGCTGGCGCTCATCAATA priA Antisense Primer 20 CGCGTGAAAACGGTTACAGA prlC-EGFP-Sense 87 GGTCGTGAACCGCAGCTGGATGCGATGCTGGAGCATTACGGCATTAAGGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG prlC-EGFP-AntiSense 72 CCGTCTCCGGTGCCTGTTTCATCAATTAAGCAGATTTTCACTGAATGATCATAGTGAACCTCTTCGAGGGAC prlC Sense Primer 21 TCAGAAGAGCCGATGGATCTG prlC Antisense Primer 23 ATCAGGTTGTCTTCATCGTGCTC prmA-EGFP-Sense 87 GACCCGGTCGTGGAAAAAGAAGAGTGGTGCCGTATTACCGGTCGTAAGAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG prmA-EGFP-AntiSense 72 TGAGCTTGCTCGCACTTCGCCCCGCGTCACCCTACGGCGATGCGAAGGTTATAGTGAACCTCTTCGAGGGAC prmA Sense Primer 23 GTGAAGCTTATGCCGATAGCTTC prmA AntiSense Primer 20 AATCGGCAAATTTACGTGCC proA-EGFP-Sense 87 GCACTGACCACTTACAAGTGGATCGGCATTGGTGATTACACCATTCGTGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG proA-EGFP-AntiSense 72 CAATGGCCTTGTGAATCAAATGGCTACTTTTGCATCACCCGGTTTTATTTATAGTGAACCTCTTCGAGGGAC proA Sense Primer 22 GGCGGTAAGCACACAAAAACTC proA Antisense Primer 23 GACAAGGTTAAAACTAACCGGGC proB-EGFP-Sense 87 GGATATGAATACGGCCCGGTTGCCGTTCACCGTGATGACATGATTACCCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG proB-EGFP-AntiSense 72 AGGCTTGCTTCGCGGCAATGCCCATTTGTTCCAGCATCAGCCTGCTCCTTATAGTGAACCTCTTCGAGGGAC proB Sense Primer 22 CACTCGCAAGAAATTGATGCAA proB Antisense Primer 21 GGCTGGAGAGTTGCGCTAATT proC-EGFP-Sense 87 ATCGAAGCGATGACGAAGTGTATGGAAAAATCAGAAAAACTCAGCAAATCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG proC-EGFP-AntiSense 72 CCGGACGTAACCGCACCGAAGTGGCGGCCTGACGTCCGGCGAAAGTCATCATAGTGAACCTCTTCGAGGGAC proC Sense Primer 22 ACTGGAAGAGAAAGGCTTCCGT proC Antisense Primer 21 TTTGGCGCTTTACAAAGCAAA proS-EGFP-Sense 87 TTAATTAAGACTGGTGACATCGTCGAATATCTGGTGAAACAGATTAAAGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 201 proS-EGFP-AntiSense 72 ACAATCCATGTAAAAAAAGGGCCCTGAAATTCAGGACCCTTTCTGGCATCATAGTGAACCTCTTCGAGGGAC proS Sense Primer 23 AATATAAATATCGTCGCAACGGC proS Antisense Primer 22 GCAGTACGCCTTTGGTTTATCC prS-EGFP-Sense 87 GCGATTCGTCGTATCAGCAACGAAGAATCGATCTCTGCCATGTTCGAACACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG prS-EGFP-AntiSense 72 ATTACAGACAAAAAAACCCGCCGCAGCGGGTCTTTGAGCCGGGTTCGATTATAGTGAACCTCTTCGAGGGAC prS Sense Primer 21 CGTGCGTACTCTGACCCTGTC prS Antisense Primer 24 GTGAAGGAGGCATAGGTCATACAA psD-EGFP-Sense 87 GAAGCAGAACACGACGCCAGCCCATTGGTTGACGACAAAAAAGACCAGGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG psD-EGFP-AntiSense 72 CATCAGAAAAGTGATAATCAGGCGCACGTCAGCGTTTCCTTTGATGGATTATAGTGAACCTCTTCGAGGGAC psD Sense Primer 20 TTTGTTACGCCAGACGCTGA psD Antisense Primer 26 AGTTCCTGAGTGATTTGTTTGCTATC ptH-EGFP-Sense 87 ACAGATGGCTTGACCAAAGCAACGAACCGATTGCACGCCTTTAAAGCGCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ptH-EGFP-AntiSense 72 CTTTGCCTATTATACACGGCACTCGGCAAAAATGCCGCAGACAACGACTTATAGTGAACCTCTTCGAGGGAC ptH Sense Primer 21 TGATGAAGCCATTGACGAAGC ptH Antisense Primer 20 CCGCATTTGAATCCCATGAT purA-EGFP-Sense 87 ACCGGTCCGGATCGTACTGAAACCATGATTCTGCGCGACCCGTTCGACGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purA-EGFP-AntiSense 72 ATCACACTGTTCGCCCGGCAGGCAAAATATCTGCCAGGCGTACCAGAATTATAGTGAACCTCTTCGAGGGAC purA Sense Primer 20 AGCTGACTGGTGTGCCGATC purA Antisense Primer 21 TAATGGCTTACCCGACACAGC purB-EGFP-Sense 87 ACGCCGGCTAACTATATTGGTCGAGCTATCACGATGGTTGATGAGCTGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purB-EGFP-AntiSense 72 CTTAATAAGCAGGCCGGACAGCATCGCCATCCGGCACTGATACGAGGTTTATAGTGAACCTCTTCGAGGGAC purB Sense Primer 22 GCGTTGCCAGAAGAAGAGAAAG purB Antisense Primer 26 TATGGGGGTAAACATTAAATAAACCA purD-EGFP-Sense 87 TGCTTCTGCCGGAAAGATATCGGCTGGCGCGCTATCGAACGCGAGCAGAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purD-EGFP-AntiSense 72 CCGTCAGTTAGGGATCACGCGCAAAACGCTATTGGCAAAACTGTCGCGTTATAGTGAACCTCTTCGAGGGAC purD Sense Primer 24 TATGCCTTAATGACCGATATCCAC purD Antisense Primer 24 GTGGAAGTGGAAAAAGAGGTGATT purE-EGFP-Sense 87 AAAGCCCAGACCGACGAAGTGCTGGAAAACCCGGACCCGCGAGGTGCGGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 202 purE-EGFP-AntiSense 72 GACGCAGCATACGGCCTAACTGCCCGTTACCGAGGACGCAAACCTGTTTCATAGTGAACCTCTTCGAGGGAC purE Sense Primer 21 AAGAACTGCACCAGCGTCTGA purE Antisense Primer 22 AGACAGCAATGCCTAACGGTTC purF-EGFP-Sense 87 GCAGTGCAACGTCAGAACGAAGTGGAAAATCTCGAAATGCATAACGAAGGACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purF-EGFP-AntiSense 72 GCGGGAGTTGCAAGTCAGGGTGCCAGACCGGCACCCTCAGCGAAGGCATCATAGTGAACCTCTTCGAGGGAC purF Sense Primer 22 ACGTTACGTAATGATGACGCCA purF Antisense Primer 19 TGCAGGCAGACTTTGCAGG purH-EGFP-Sense 87 GACGAGCACGGTATTGCGATGCTCTTCACCGACATGCGCCACTTCCGCCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purH-EGFP-AntiSense 72 GCTCGCGCCCGCCGTTACCAATCACTAATACTTTCATCTATTGCTCCATTATAGTGAACCTCTTCGAGGGAC purH Sense Primer 23 TATCCGTGATGACGAAGTGATTG purH Antisense Primer 24 AGCAACAAAAACAGTCTCAACCAG purK-EGFP-Sense 87 CTGCCGCCGGAATATGCCAGCGGCGTGATTTGGGCGCAGAGTAAGTTCGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purK-EGFP-AntiSense 72 AGGCCGGATAAGGCGTTTACGCCGCATCCGGCAAGAATAGAGCACCAGTTATAGTGAACCTCTTCGAGGGAC purK Sense Primer 20 CGACACATCGCGTCTGACTG purK Antisense Primer 19 GGCCTACAATGGGTACCGG purL-EGFP-Sense 87 GAGGATGGCCCATGGATGCGCATTTTCCGCAATGCGCGTAAGCAGTTGGGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purL-EGFP-AntiSense 72 TTGGTGCTTAATGCCGCATCCGGGCTGCAAAACCAATGGGCTGACGACTTATAGTGAACCTCTTCGAGGGAC purL Sense Primer 19 CTCCTGGCATCCGGAAAAC purL Antisense Primer 20 TCCCGGAGTTGGGAGCTTAT purM-EGFP-Sense 87 AAAATCGGTATCATCAAAGCCTCTGATTCCGAACAACGCGTGGTTATCGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purM-EGFP-AntiSense 72 TTGCCTGTAAATTACTTCCGTTGCCGGAAATAAGCACCACAATATTCATTATAGTGAACCTCTTCGAGGGAC purM Sense Primer 19 TCCGGAAGTGGACAAAGCC purM Antisense Primer 22 TTTGTTGGTTTTACAGGCGTCA purN-EGFP-Sense 87 GCGTGGCTGGATGGTCAACGTCTGCCGCCGCAGGGCTACGCTGCCGACGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG purN-EGFP-AntiSense 72 GAATTGAAAAACGCCAGCGGCAGAGCTGGCGCTTTAATTACGGGGGTATTATAGTGAACCTCTTCGAGGGAC purN Sense Primer 21 GGTCGTCTGAAAATGCACGAA purN Antisense Primer 21 CTGCTGGAGCTTGCGATTTAC pykA-EGFP-Sense 87 GTGATGAGTACCGTGGGTTCTACTAATACCACGCGTATTTTAACGGTAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 203 pykA-EGFP-AntiSense 72 TTGAACTGTAGGCCGGATGTGGCGTTTTCGCCGCATCCGGCAACGTACTTATAGTGAACCTCTTCGAGGGAC pykA Sense Primer 22 TTGATGTCTGGTGACCTGGTGA pykA Antisense Primer 24 CGCCTGATGATAAGTTCAAGTTTG pyrD-EGFP-Sense 87 GGTTTTATTTTTAAAGGTCCGCCGCTGATTAAAGAAATCGTTACCCATATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pyrD-EGFP-AntiSense 72 TGAAAATAAAGCCCTGATTATCTTATAAAGAGGAATTCGAAGAAATAATTATAGTGAACCTCTTCGAGGGAC pyrD Sense Primer 22 GTGCCTCACTGGTGCAAATTTA pyrD Antisense Primer 25 CAACAGCGATGAATATAAAACAACC pyrE-EGFP-Sense 87 ATGGCGGAACATCTGGCGGCGGTTAAGGCCTATCGCGAAGAGTTTGGCGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pyrE-EGFP-AntiSense 72 CAGTTGCAGTAATATGACGCCGGATGACTTTTCATCCGGCGAGTTTCTTTATAGTGAACCTCTTCGAGGGAC pyrE Sense Primer 23 CCTGATTGCTTACCTGGAAGAGA pyrE Antisense Primer 22 TTGTCCGCAGCGAATTTAAATA pryF-EGFP-Sense 87 GATCCAGCGCAGACGCTGAAAGCGATCAACGCCTCTTTACAGCGGAGTGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pyrF-EGFP-AntiSense 72 TACGTCCGGTTTCCGTTGAGTAGACCAGACGGCTGTTGGAATCACTCATCATAGTGAACCTCTTCGAGGGAC pyrF Sense Primer 24 GTTGTCGGCTGGTGTTGATTATAT pyrF Antisense Primer 19 CACCACACCGTCGCCTTTA pyrG-EGFP-Sense 87 TTTGCAGGCTTTGTGAAAGCCGCCAGCGAGTTCCAGAAACGTCAGGCGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG pyrG-EGFP-AntiSense 72 ACAAACAACGCGTACCCAGGGTACGCGTTGCCGCTCTAACTTTTTTACTTATAGTGAACCTCTTCGAGGGAC pyrG Sense Primer 20 ACTCCACGTGATGGTCACCC pyrG Antisense Primer 25 GGACATTAGGTTTTCCTCAAGTCAC queA-EGFP-Sense 87 GCGATGTTTATCACGTACAATCCGCAGGCAATTAATGAGCGCGTCGGGGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG queA-EGFP-AntiSense 72 CCACTACGTCAGAAAAACAGTCCAACGTTTTAAACCAGCGCCGCGGAATTATAGTGAACCTCTTCGAGGGAC queA Sense Primer 22 CGCCTATAAAGCAGCGGTAGAA queA Antisense Primer 20 GTCGGTGGTGTCCAGTTCAA radA-EGFP-Sense 87 ATTTTTGGCGTTAAAAAACTCTCCGACGCGCTTAGCGTGTTCGACGACTTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG radA-EGFP-AntiSense 72 CAGTTTTCAGGTAATCAAATGACGACATATCTCCCTCCGTATATCTCATTATAGTGAACCTCTTCGAGGGAC radA Sense Primer 21 AGCGGTCAGGAACGAATCTCT radA Antisense Primer 20 GCTACCTGCTGTAGCGTGCA rbfA-EGFP-Sense 87 GTCAAACATGACGAAGAACGTCGTGTTAACCCGGACGACAGCAAGGAGGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 204 rbfA-EGFP-AntiSense 72 GCAACAAAACGCCGTTAATGTCGCGACCGCGACGACGAGGACGACTCATTATAGTGAACCTCTTCGAGGGAC rbfA Sense Primer 19 TGCGCATGTCAAACCTGGT rbfA Antisense Primer 22 TTGCTGGACATACCCTGAGGTT recA-EGFP-Sense 87 GATTTCTCTGTAGATGATAGCGAAGGCGTAGCAGAAACTAACGAAGATTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recA-EGFP-AntiSense 72 AAAAGCAAAAGGGCCGCAGATGCGACCCTTGTGTATCAAACAAGACGATTATAGTGAACCTCTTCGAGGGAC recA Sense Primer 19 TGCTGAGCAACCCGAACTC recA Antisense Primer 21 CGACGGGATGTTGATTCTGTC recC-EGFP-Sense 87 GTTGAACAGTCGCAACGTTTCCTGTTACCGCTGTTTCGCTTTAATCAGTCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recC-EGFP-AntiSense 72 TGCGCATCATAAAGTAAGCGGATAGATTGCGCAATTTTTATACAGCACTCATAGTGAACCTCTTCGAGGGAC recC Sense Primer 22 TAACACCAGAGACAATGGAGGC recC Antisense Primer 19 GTGCTGCACGAGTCAGCCT recD-EGFP-Sense 87 ACTCGTACTGAGCGGCGCAGTGGTCTGGCGGCGTTGTTTAGTTCACGGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recD-EGFP-AntiSense 72 TCGGATGCGACATGCGTAACACTCGTACGTCGCATCCGGCAATTACGTTTATAGTGAACCTCTTCGAGGGAC recD Sense Primer 20 CGCATATTAAGTGCGGCAAT recD Antisense Prime 19 GGCGGATTTAGGGTAAGCG recF-EGFP-Sense 87 TCGGACGAAAATTCGAAGATGTTTACCGTGGAAAAGGGTAAAATAACGGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recF-EGFP-AntiSense 72 GAGTCATAAGAATTCGACATCAACGTTTCTCGCTCATTTATACTTGGGTTATAGTGAACCTCTTCGAGGGAC recF Sense Primer 19 ACAGGTCTTTGTCAGCGCG recF Antisense Primer 23 GCCCTTTCAGGACTTTGATACTG recG-EGFP-Sense 87 AAAGCCCTGATAGAACGCTGGATGCCGGAGACGGAACGTTACTCGAATGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recG-EGFP-AntiSense 72 GCAGGAAGGTAGGGTAACCTGAAATGGCGGTCTTCTCACTGCCGCCTTTTATAGTGAACCTCTTCGAGGGAC recG Sense Primer 21 ACGAACGTTACCCACAACAGG recG Antisense Primer 21 GTAGGGTGATGAATCGCATCC recJ-EGFP-Sense 87 TTTCGCGGCAACCGCAGCCTGCAAATTATCATCGACAATATCTGGCCAATTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recJ-EGFP-AntiSense 72 GAGCGGGATTGTACCCAATCCACGCTCTTTTTTATAGAGAAGATGACGCTATAGTGAACCTCTTCGAGGGAC recJ Sense Primer 25 CAACTGGCTTATAAGCTCGATATCA recJ Antisense Primer 24 TGAGCTAGTCAAAATGCGGTGATA recN-EGFP-Sense 87 AGTGAAGTCACACGTAATACACTGGCGAATGCGAAAGAACTGCTTGCAGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 205 recN-EGFP-AntiSense 72 TCTTACGGCGTTTTGCTGTTTACTCTGACCGTGAAGCAGGAAAAAAGTTTATAGTGAACCTCTTCGAGGGAC recN Sense Primer 19 AAGCGCGGTTACAAGAGCT recN Antisense Primer 26 TCATCACTTTAAAACCTTTTGCTTTC recO-EGFP-AntiSense 72 TATGGTCAATGTTGACGCCTAACAGTAATTCAGCCATGACAATCCTCATCATAGTGAACCTCTTCGAGGGAC recO-EGFP-Sense 87 GAACTGTTCCGGCAGTTTATGCCTAAGCGAACGGTGAAAACACATTATGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recO Sense Primer 20 TTAAGCCGTATCTTGGCGGT recO Antisense Prime 18 CACCGGATCCGGGTAAGC recQ-EGFP-Sense 87 AAACCGTTTATGGCGCTGATTCGTGCGCATGTTGATGGCGATGACGAAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recQ-EGFP-AntiSense 72 GGGAAACGTTTACGGAGTCTTCATACTGGCACTTTTTTATGCTGCTGACTATAGTGAACCTCTTCGAGGGAC recQ Sense Primer 23 TGATTGAGATGGCTGAACAGATG recQ Antisense Primer 23 CATCAACATACATTTGACTCGCG recR-EGFP-Sense 87 GACGGCACCACGTTGTCACACTCCCTTGCCGGGCGTCATAAGATTCGTTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG recR-EGFP-AntiSense 72 AAGGGAGAATAATTTCAAGCGAGAGCAGGTGATCCTGCTCTCGTTTGCTTATAGTGAACCTCTTCGAGGGAC recR Sense Primer 18 AATCGCTCATGGCGTTCC recR Antisense Primer 22 AAACAGGATGTGGGAGAGATGG relA-EGFP-Sense 87 AAACTCAACCAGGTGCCGGATGTTATCGACGCGCGTCGGTTGCACGGGAGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG relA-EGFP-AntiSense 72 AGATACAGTATATATCAATCTACATTGTAGATACGAGCAAATTTCGGCCTATAGTGAACCTCTTCGAGGGAC relA Sense Primer 22 ACCATCGACATGACCATTGAGA relA Antisense Primer 22 CCTTTCCTCAAACCGCTATCAT rep-EGFP-Sense 87 CAAAGCCATCTGGCGAATCTGAAAGCGATGATGGCGGCAAAACGAGGGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rep-EGFP-AntiSense 72 AATGAGTAAGTGCCGGATGCGATGCTGACGCATCTTTTCCGGCCTTGATTATAGTGAACCTCTTCGAGGGAC rep Sense Primer 20 AACAGGAGCGCAAAGTGGTC rep Antisense Primer 23 ATTATTGCTCAGGAAAGCCAGTG rhO-EGFP-Sense 87 CTGGCAATGACCAAGACCAATGACGATTTCTTCGAAATGATGAAACGCTCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rhO-EGFP-AntiSense 72 AAAGCAAAACGCCACGTAAACACGTGGCGTTTTTGGCATAAGACAAATTTATAGTGAACCTCTTCGAGGGAC rhO Sense Primer 22 AAATCGATGCAATGGAATTCCT rhO Antisense Primer 21 GCGCGGCATTAAGATTACAGA ribC-EGFP-Sense 87 CGTGTGCTGGCGGCACGAGAAAATGCCATGAATCAACCAGGCACAGAAGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 206 ribC-EGFP-AntiSense 72 GCTCGCTAAAGGCTATTCTATCGCCCCCTCTCCGGGGGCGATTTCAGATCATAGTGAACCTCTTCGAGGGAC ribC Sense Primer 24 CAAACTCAGGCAGTGGTAGATACG ribC Antisense Primer 19 CGGTGTTGAAAACGGCCTT ribD-EGFP-Sense 87 AAAGAGATACGTCATGTAGGCCCGGATGTTTGCCTGCATTTAGTGGGTGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ribD-EGFP-AntiSense 72 GATTTTAGCATAATATTTCGTGCGCTGCTTCCCTTTCGAGCCGGGAGATCATAGTGAACCTCTTCGAGGGAC ribD Sense Primer 23 GCACCTAAACTATTAGGCAGCGA ribD Antisense Primer 25 GCAACGTTAGCTTCAATAATGTTCA ribE-EGFP-Sense 87 GCTGCACTGACCGCGCTTGAAATGATTAATGTATTGAAAGCCATCAAGGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ribE-EGFP-AntiSense 72 CGAGCGCGGCGACGAGCAGCAGGTTTCACGGATTTCCCCTTACTAATTTCATAGTGAACCTCTTCGAGGGAC ribE Sense Primer 19 GCACCAAAGCTGGCAACAA ribE Antisense Primer 20 ATCAGCGATGTCGTTCTGGG ribF-EGFP-Sense 87 GCGCGTGATGAATTAACCGCCCGCGAATTTTTTGGGCTAACAAAACCGGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ribF-EGFP-AntiSense 72 ACTCATCAGATTCTCGGTTCCGTATTTCGGTTTGATTACATAACAGGCTTATAGTGAACCTCTTCGAGGGAC ribF Sense Primer 19 CGTCGCTGGACGAACTGAA ribF Antisense Primer 19 TTCCGGCAAATTCAGGGTT rimI-EGFP-Sense 87 ACCACGGACGGTCGCGAAGACGCCATCATCATGGCGTTGCCAATCAGTATGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rimI-EGFP-AntiSense 72 CATCGGCATCAAAGAAAATCCAGTCCCACTTCATTATTCCACCTTGTATTATAGTGAACCTCTTCGAGGGAC rimI Sense Primer 21 GACGATTCGCCGCAATTACTA rimI Antisense Primer 24 CGGTGAATGAGTCAAAGGTAAACA rimM-EGFP-Sense 87 AAAGTCGATCTCACTACTCGTTCAATCGAAGTAGATTGGGATCCTGGTTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rimM-EGFP-AntiSense 72 AATTATGCCAATCCACATAGCGCCGTCTTTTACCGTTTATCCGGTGGTTTATAGTGAACCTCTTCGAGGGAC rimM Sense Primer 20 ACGTCTCGTACCGTTCCTCG rimM Antisense Primer 19 TAATCGGTAATTGCGCGGA rluC-EGFP-Sense 87 CCGATGGATGAAGGTTTGAAGCGTTGTTTGCAAAAGCTGCGTAACGCGCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rluC-EGFP-AntiSense 72 AAACGCCTTATCAGGCCAACCGCCCATTATCAGGTTTATATGCTTGTATTATAGTGAACCTCTTCGAGGGAC rluC Sense Primer 21 TGAGGTGATGCGTATCGAAGC rluC Antisense Primer 22 AAAACCCGCTGATGGGATAGTT rluD-EGFP-Sense 87 GAGGTGATGCGCGCCGATTTCGAAGAACATAAGGATGAAGTGGACTGGTTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 207 rluD-EGFP-AntiSense 72 AGGCCGCAACACCTTTTGGCTGCGGCCACTGCGGGACAATCAGCTTACTCATAGTGAACCTCTTCGAGGGAC rluD Sense Primer 22 TCCACAAGATATGGTGGAGCTG rluD Antisense Primer 23 ACCGAGGTTGAGTGAGTCATACG rnC-EGFP-Sense 87 AAGGCTGAGCAGGCTGCCGCCGAACAGGCGTTGAAAAAACTGGAGCTGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rnC-EGFP-AntiSense 72 TCGGACGTCCGACGATGGCAATAAATCCGCAGTAACTTTTATCGATGCTCATAGTGAACCTCTTCGAGGGAC rnC Sense Primer 23 TTACTATCCACTGCCAGGTCAGC rnC Antisense Primer 22 TGTTCAACAATGTGGATTTGCC rnD-EGFP-Sense 87 GGTGAGCTGATGGCGGAAGCATTACACAATTTATTGCAGGAATATCCGCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rnD-EGFP-AntiSense 72 CGAAGCCGGATGTGGCGCTGAGCGCGCCCAGTCCGGCTTCGGAAGATTTTATAGTGAACCTCTTCGAGGGAC rnD Sense Primer 20 AGAACAATTTGCCGGAGCTG rnD AntiSense Primer 20 GGTAAACGCCTGGTGTTGGA rnE-EGFP-Sense 87 ACGGCAACACATCATGCCTCTGCCGCTCCTGCGCGTCCGCAACCTGTTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rnE-EGFP-AntiSense 72 TAAAAAAGCCCTGGCAGTTACCAGGGCTTGATTACTTTGAGCTAATTATTATAGTGAACCTCTTCGAGGGAC rnE Sense Primer 19 TAAAGGTGCCGCAGGTGGT rnE Antisense Primer 22 ATTGTTGGTTAGCAAGGATGCC rnhA-EGFP-Sense 87 GCCGCGGCGATGAATCCCACACTGGAAGATACAGGCTACCAAGTTGAAGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rnhA-EGFP-AntiSense 72 AATTCGCCAGGCGGTTGGAGCCACCCGGCAATGTCGTAAACCACAGGCTTATAGTGAACCTCTTCGAGGGAC rnhA Sense Primer 23 GAAAACGAACGCTGTGATGAACT rnhA Antisense Primer 23 AACGCTAAATCCGATGAAACAGA rnhB-EGFP-Sense 87 CACCATCGGCGCAGCTTTGGGCCTGTCAAACGCGCACTGGGACTTGCGTCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rnhB-EGFP-AntiSense 71 GGTTCAGACATCTTCAGATTCCGGTTTACTTAATCTCGACACAAGAATCATAGTGAACCTCTTCGAGGGAC rnhB Sense Primer 21 GTACCCAACCGCTTTTCATCT rnhB Antisense Primer 21 TCATCGAGTAGTCGCTGTGCA rnpA-EGFP-Sense 87 GAAGCGTTGGAAAAATTATGGCGCCGCCACTGTCGCCTGGCTCGCGGGTCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rnpA-EGFP-AntiSense 72 GCCCGAGTAGCGGACTAATCAGGCGTTGATAGACCCGAATGAGGGCTATCATAGTGAACCTCTTCGAGGGAC rnpA Sense Primer 21 CTATGGATTTCGTGGTGGTGG rnpA Antisense Primer 23 CTCAATTCCGTAGCTTGAACAGG rnR-EGFP-Sense 87 ATAGCTGCAGCGACCAAAGCGAAGCGTGCGGCGAAGAAAAAAGTGGCAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 208 rnR-EGFP-AntiSense 72 GCGCATTTTGTCAGCAATCTAACCCTCTTCTTTTAAAGAGGGTATTGATCATAGTGAACCTCTTCGAGGGAC rnR Sense Primer 20 CGAGAAAAGCGAAAAAGCCA rnR Antisense Primer 23 GAATTTGCGTGATTATGTAGGCC rnT-EGFP-Sense 87 AACCGCTGGAAACGTCTGGGAGGCTGGCCGCTATCTGCCGCCGAAGAGGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rnT-EGFP-AntiSense 72 CTCACTATAGATGCCTGAATCATTGCATGTCATCAGGCATCGACTCGATTATAGTGAACCTCTTCGAGGGAC rnT Sense Primer 21 GCACTGCTGTGCTGTTTTGTG rnT Antisense Primer 22 GTGAATATCATGCAGGATGCGT rpE-EGFP-Sense 87 AAAAAAGTCATTGATGAAATGCGCAGTGAACTGGCAAAGGTAAGTCATGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpE-EGFP-AntiSense 72 ACCAGCGTACCATCAAGATCAAAAGCGACGCCGCGAATATCTTCAAACTTATAGTGAACCTCTTCGAGGGAC rpE Sense Primer 20 GCAATCTTCGACCAGCCAGA rpE Antisense Primer 21 AGCAGCAAGACCAGGAGCACT rpH-EGFP-Sense 87 GAAGAGCTACTCATCTTGTTGGCTCTGGCCCGAGGGGAATCGAATCCATTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpH-EGFP-AntiSense 72 TCATCAGTCGCCTTAAAAATCAGTTTGCCAGCGCCGCCTTCTGCGTCGCTATAGTGAACCTCTTCGAGGGAC rpH Sense Primer 23 GACATGAACGTAGTGATGACCGA rpH Antisense Primer 20 TCATGCCTTCGCTCCTCATC rpiA-EGFP-Sense 87 GCGGACGTTGCGCTGATTGGCACACCTGACGGTGTCAAAACCATTGTGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpiA-EGFP-AntiSense 72 AAATTTAATGAAGAGAATTTTTTTAACGGGGGAGGTTCCCCCGTCAGATCATAGTGAACCTCTTCGAGGGAC rpiA Sense Primer 22 GGTGACTGTTGGCTTGTTTGCT rpiA Antisense Primer 22 GCCTGGTAAAAGCGTGACACAT rplA-EGFP-Sense 87 ATGGGTGCAGGTGTTGCAGTTGACCAGGCTGGCCTGAGCGCTTCTGTAAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplA-EGFP-AntiSense 72 ATACGTGGGGGTAAGATTGTAGACAAAATCACCGCCCACGTAAAGGCATTATAGTGAACCTCTTCGAGGGAC rplA Sense Primer 24 GGCGTGTACATCAAGAAAGTTAGC rplA Antisense Primer 20 GCTCCTGGCGACTCACTCAG rplB-EGFP-Sense 87 ACCCGCAGCAACAAGCGTACTGATAAATTCATCGTACGTCGCCGTAGCAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplB-EGFP-AntiSense 72 ATAAAAGGACCTTTCTTGAGAGAACGTGGCATGGCTTATCCTCTAAAATTATAGTGAACCTCTTCGAGGGAC rplB Sense Primer 23 GCGTTCAGACCAAAGGTAAGAAG rplB Antisense Primer 23 CTACCTTCTTCAGCAAGTGCAGG rplC-EGFP-Sense 87 GTCCCGGGTGCAACCGGTAGCGACCTGATCGTTAAACCAGCTGTGAAGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 209 rplC-EGFP-AntiSense 72 GTCAGCGCGCTCTGCGCGTCTTTCAATACTAATTCCATTGCTATCTCCTTATAGTGAACCTCTTCGAGGGAC rplC Sense Primer 22 CAACCTGCTGCTGGTTAAAGGT rplC Antisense Primer 22 ATCACGACCGAAGGTAGTTTCG rplD-EGFP-Sense 87 AAAGTCGTAATGACTGCTGATGCTGTTAAGCAAGTTGAGGAGATGCTGGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplD-EGFP-AntiSense 72 CAGAAACGTGCGGTGCACGCAGCACCTTCAGCAGACGTTCTTCACGAATCATAGTGAACCTCTTCGAGGGAC rplD Sense Primer 21 GTTGACGTACGCGATGCAACT rplD Antisense Primer 23 GTCGCGTCTTTAGCAACTTTGAG rplE-EGFP-Sense 87 GACGAAGAAGGCCGCGCTCTGCTGGCTGCCTTTGACTTCCCGTTCCGCAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplE-EGFP-AntiSense 72 TTACTTCGCGTGCTTTCATTGATTGCTTAGCCATTTAGTAACCCTACCTTATAGTGAACCTCTTCGAGGGAC rplE Sense Primer 23 CGTTCGTGGTTTGGATATTACCA rplE Antisense Primer 19 GCGCGTTTCGCGAAGTATT rplF-EGFP-Sense 87 GGTGTTCGTTACGCCGACGAAGTCGTGCGTACCAAAGAGGCTAAGAAGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplF-EGFP-AntiSense 72 GCGGGTCGCACGACGGATACGAGCAGATTTCTTATCCATAGTGTTACCTTATAGTGAACCTCTTCGAGGGAC rplF Sense Primer 19 CCTACCGTCGTCCTGAGCC rplF Antisense Primer 20 CGATGTACCACCAGGCGAGT rplI-EGFP-Sense 87 CAGGTTCACAGCGAAGTATTCGCGAAAGTGATCGTAAACGTAGTAGCTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplI-EGFP-AntiSense 72 AAAACGCCGACCAATGGTCGGCGTTTTTACGTCTCGTTGAATAACGAATTATAGTGAACCTCTTCGAGGGAC rplI Sense Primer 21 GTACCACTGGCGAACACGAAG rplI Antisense Primer 25 GAGAATGGTGAATGGATCTACTTGC rplJ-EGFP-Sense 87 GGCAAACTGGTTCGTACTCTGGCTGCTGTACGCGATGCGAAAGAAGCTGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplJ-EGFP-AntiSense 72 TCAGAATAAGTTTATACGTAAGCGAATGCGTTAAAAAGATAACTGCGATTATAGTGAACCTCTTCGAGGGAC rplJ Sense Primer 21 GCAACCATGAAAGAAGCTTCG rplJ Antisense Primer 23 TGCAACTGCTTCAATGATTTGAT rplK-EGFP-Sense 87 ACTCGCTCCATCGAAGGTACTGCACGTTCCATGGGCCTGGTAGTGGAGGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplK-EGFP-AntiSense 72 AACTTTCTCGCGGATAACACGCATGCGCTTGGTCAGTTTAGCCATTTCTTATAGTGAACCTCTTCGAGGGAC rplK Sense Primer 19 CAGACCAAAGCTGCCGACA rplK Antisense Primer 24 CGATAGCTTCGTTGATGTCGTACT rplL-EGFP-Sense 87 GAAGCACTGAAAAAAGCTCTGGAAGAAGCTGGCGCTGAAGTTGAAGTTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 210 rplL-EGFP-AntiSense 72 TCACCAGCCATCAGCCTGATTTCTCAGGCTGCAACCGGAAGGGTTGGCTTATAGTGAACCTCTTCGAGGGAC rplL Sense Primer 20 GCTGCTCTGAAAGAAGGCGT rplL Antisense Primer 18 GCGCAAAAAGGCTGGTGA rplM-EGFP-Sense 87 GCGGGTAACGAGCACAACCACGCGGCACAGCAACCGCAAGTTCTTGACATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplM-EGFP-AntiSense 72 GCGACCAGTGCCGTAGTATTGATTTTCAGCCATTGCCTATAATCCCGATTATAGTGAACCTCTTCGAGGGAC rplM Sense Primer 20 AAAGGCATGTTGCCAAAAGG rplM Antisense Primer 20 TTTGATGAAAACGCGAGCTG rplN-EGFP-Sense 87 CTTCGTAGTGAGAAGTTCATGAAAATTATCTCTCTGGCACCAGAAGTACTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplN-EGFP-AntiSense 72 ACGATAACTTCGTCATCACGACGGATTTTCGCTGCCATGATTCGCTCCTTATAGTGAACCTCTTCGAGGGAC rplN Sense Primer 22 CAGCCTATCGGTACGCGTATTT rplN Antisense Primer 23 CGCGTTTACCTTTATCTTTACCG rplO-EGFP-Sense 87 ACTAAAGGCGCTCGTGCTGCTATCGAAGCTGCTGGCGGTAAAATCGAGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplO-EGFP-AntiSense 72 CCTTTGGCACTTTGAAAATCTAATCCCGGTTGTTTAGCCATCTGCTACTTATAGTGAACCTCTTCGAGGGAC rplO Sense Primer 20 CGAGTTCGCGAAAGTGATCC rplO Antisense Primer 21 ACAGCAGTCTGCGTTTCAGCT rplP-EGFP-Sense 87 GCAGCAGCGAAACTGCCGATTAAAACCACCTTTGTAACTAAGACGGTGATGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplP-EGFP-AntiSense 72 CGGTGTTCAGCTCTTCAACGCTCTTCTCACGCAGCTCTTTTGCTTTCATTATAGTGAACCTCTTCGAGGGAC rplP Sense Primer 23 CTGTATGAAATGGACGGTGTTCC rplP Antisense Primer 22 CATACGCAGGTTGAACTGCTCA rplQ-EGFP-Sense 87 GCTTACATCGAGCTGGTTGATCGTTCAGAGAAAGCAGAAGCTGCTGCAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplQ-EGFP-AntiSense 72 CGGGTATAAAAAAACCCGCCGGGGCGGGTTTTTTTACGTTGCTTCAGATTATAGTGAACCTCTTCGAGGGAC rplQ Sense Primer 23 CCGGTGGTTACACTCGTATTCTG rplQ Antisense Primer 24 CCACATAAATACTCCAGGGGATGA rplR-EGFP-Sense 87 GGTCGTGTCCAGGCACTGGCAGATGCTGCCCGTGAAGCTGGCCTTCAGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplR-EGFP-AntiSense 72 GCAGTTCGCCAGCTTGTTTTTCGATGTGAGCCATCTTACACCTCTACCTTATAGTGAACCTCTTCGAGGGAC rplR Sense Primer 22 GATGTATCCTTTGACCGTTCCG rplR Antisense Primer 19 CGGTTTACCGCGATCAGCT rplS-EGFP-Sense 87 TACCTGCGTGAGCGTACTGGTAAGGCTGCTCGTATCAAAGAGCGTCTTAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 211 rplS-EGFP-AntiSense 72 CAGCCAATTGGCCAGCCCTTCTTAACAGGATGTCGCTTAAGCGAAATCTTATAGTGAACCTCTTCGAGGGAC rplS Sense Primer 19 AACGTCGTGGTGCTGTTCG rplS Antisense Primer 19 GCGTCGCAGTGGTGATTACTAC rplT-EGFP-Sense 87 GACAAAGTAGCGTTCACCGCTCTGGTTGAAAAAGCGAAAGCAGCTCTGGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplT-EGFP-AntiSense 72 ATGGCGTTGAAACGAAAAGAGGGAGACTAGCTCCCTCTTTCAACTGGCTTATAGTGAACCTCTTCGAGGGAC rplT Sense Primer 20 CGACCGTAAGATCCTGGCTG rplT Antisense Primer 22 AAGGCTACGGCGATAAAAGTCA rplU-EGFP-Sense 87 CAGGGCCATCGTCAGTGGTTCACTGATGTGAAAATTACTGGCATCAGCGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplU-EGFP-AntiSense 72 TGGAGCCGCCAGCCTTTTTATGTGCCATTTGAAATCTCTCCTCAGGTCTTATAGTGAACCTCTTCGAGGGAC rplU Sense Primer 20 CGGTCGTGGCGAGAAAGTTA rplU Antisense Primer 19 AGCTTCTGAATCGCGACCG rplV-EGFP-Sense 87 GATCGCATCCTGAAGCGCACCAGCCACATCACTGTGGTTGTGTCCGATCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplV-EGFP-AntiSense 72 GAATACCATTAGGATGTACTTTCTGACCCATTGCTAGTCTCCAGAGTCTCATAGTGAACCTCTTCGAGGGAC rplV Sense Primer 20 AGCATGAAGCGCATTATGCC rplV Antisense Primer 20 TTTGGTGTTCGCAAACCAGG rplW-EGFP-Sense 87 TACGTCACCCTGAAAGAAGGCCAGAATCTGGACTTCGTTGGCGGCGCTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplW-EGFP-AntiSense 72 GAGATGTCGGTTTACATTTAACAACTGCCATTGTATTACTCCTCCGACTTATAGTGAACCTCTTCGAGGGAC rplW Sense Primer 21 GACAGCGTATCGGTCGTCGTA rplW Antisense Primer 22 GTGCAGCTCAGGGTTAACCACT rplX-EGFP-Sense 87 GAAGACGGTAAAAAAGTCCGTTTCTTCAAGTCTAACAGCGAAACTATCAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rplX-EGFP-AntiSense 72 CTTCGTCTTTGTAGTAATCATGCAGTTTCGCCATCGTACTACTCCAAATTATAGTGAACCTCTTCGAGGGAC rplX Sense Primer 22 GGCTGACCGTGTAGGCTTTAGA rplX Antisense Primer 19 CCCGAGGGACTTGCATGAC rpmA-EGFP-Sense 87 TTCGAAGTTAAAGGCCCGAAAAACCGTAAATTTATCAGCATCGAAGCTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmA-EGFP-AntiSense 72 CCCGCAACGTGTTGCGGGGCTTTCATCCGTTACCGGGACGCGAAAAACTTATAGTGAACCTCTTCGAGGGAC rpmA Sense Primer 24 TTGCTAAAGCAGACGGTAAAGTGA rpmA Antisense Primer 20 GGATTAACGTCGCACCATGC rpmB-EGFP-Sense 87 AAAGGCATCGATACAGTTCTGGCTGAACTGCGTGCCCGTGGCGAAAAGTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 212 rpmB-EGFP-AntiSense 72 TGATTTTCTCACGAATACCTTTAGCCATGATTTATTTCCTCTAAGTACTTATAGTGAACCTCTTCGAGGGAC rpmB Sense Primer 19 TTTTGTCACCCTGCGCGTA rpmB Antisense Primer 22 CCAGTTTTTCCGGCTTAGTACG rpmC-EGFP-Sense 87 CGTCGCGATGTCGCACGCGTTAAGACTTTACTGAACGAGAAGGCGGGTGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmC-EGFP-AntiSense 72 TTTTGTCGCTAACAACGCGACCTTGCAGAGTACGGATTTTATCGGTCATTATAGTGAACCTCTTCGAGGGAC rpmC Sense Primer 21 CCAGCTGCAACAGTCTCACCT rpmC Antisense Primer 22 CGTTCGATAGCAACAACAATGG rpmD-EGFP-Sense 87 GCTATTCGCGGTATGATCAACGCGGTTTCCTTCATGGTTAAAGTTGAGGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmD-EGFP-AntiSense 72 CGCCTTTTTGGAGCCTTCGGCCGGAGACAGAGTATTTAAACGCATCTCTTATAGTGAACCTCTTCGAGGGAC rpmD Sense Primer 20 TGCGTCGTATTGGTCACACC rpmD Antisense Primer 20 GAACCGATACCACGACCCAG rpmE-EGFP-Sense 87 GGTGGCCGTGTTGACCGCTTCAACAAGCGTTTCAACATCCCGGGCAGCAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmE-EGFP-AntiSense 72 TCAGGCACAAAAAAAGCGCCGTGCGGCGCTTTTTTCGGAAATCCGGTCTTATAGTGAACCTCTTCGAGGGAC rpmE Sense Primer 20 CACTGGCAAACAGCGTGATG rpmE Antisense Primer 23 GCTGTTTTCGTGGTAATACGACC rpmF-EGFP-Sense 87 CACCACATCACTGCCGACGGTTACTACCGCGGCCGCAAGGTCATCGCTAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmF-EGFP-AntiSense 72 AGTTGCCTGGGGAAAATCCTCACTAAGCTTCATCACGCAGATGCGTGATTATAGTGAACCTCTTCGAGGGAC rpmF Sense Primer 24 GTCACCAGCCTGTCTGTAGACAAA rpmF Antisense Primer 24 GGGTTAGACGTGTCAAGGTATCGT rpmG-EGFP-Sense 87 TTCGATCCAGTTGTTCGCCAGCACGTGATCTACAAAGAAGCGAAAATCAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmG-EGFP-AntiSense 72 ACAAAAAACCCCGCCGGAGCGAGGTTTTTTGTTACATCAAAGCGAGAATTATAGTGAACCTCTTCGAGGGAC rpmG Sense Primer 22 TAAGCCGGAAAAACTGGAACTG rpmG Antisense Primer 23 GAACAGATGCAGTCAATATGGGG rpmH-EGFP-Sense 87 GTTCTGGCACGTCGTCGTGCTAAAGGCCGCGCTCGTCTGACCGTTTCTAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmH-EGFP-AntiSense 72 CGTAACTCCCTGGGAAATGCGAGCTTAACCACTCAGGGGTTAGCTTTATTATAGTGAACCTCTTCGAGGGAC rpmH Sense Primer 22 TTCCGTGCTCGTATGGCTACTA rpmH Antisense Primer 23 CGAATGTGAATTGACTGGGAGTT rpmI-EGFP-Sense 87 ATGGTTTCCAAAGGCGATCTGGGCCTGGTAATCGCGTGCCTGCCGTACGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 213 rpmI-EGFP-AntiSense 72 GCTCTCCTGTATCTATATTCTAATTAAAAAGTTAAAAACGTTAACGGCTTATAGTGAACCTCTTCGAGGGAC rpmI Sense Primer 21 ACCTGCGTCACATTCTGACCA rpmI antisense Primer 23 AATAACACCACGTTTTACGCGAG rpmJ-EGFP-Sense 87 GTCATCCGTGTGATTTGCAGTGCCGAGCCGAAGCATAAACAGCGCCAAGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpmJ-EGFP-AntiSense 72 TAATCTAGCCAGCTCAACCCAACTTTGCAAGAAAAATATGCGAAAAAATCATAGTGAACCTCTTCGAGGGAC rpmJ Sense Primer 21 TGCCGTAACTGCAAAATCGTT rpmJ Antisense Primer 24 ACGCACAGACATACAAAAGATTGG rpoA-EGFP-Sense 87 TCTCTGGGCATGCGCCTGGAAAACTGGCCACCGGCAAGCATCGCTGACGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpoA-EGFP-AntiSense 72 TGGCGCATGACCTTATCCTTCTCAGTAAAACCTTAACCTGTGATCCGGTTATAGTGAACCTCTTCGAGGGAC rpoA Sense Primer 23 ACTGAGATTAAAGACGTGCTGGC rpoA Antisense Primer 21 CGGTTCAGTTGACGACCACTC rpoB-EGFP-Sense 87 TTGTTGAAAGAGATTCGTTCGCTGGGTATCAACATCGAACTGGAAGACGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpoB-EGFP-AntiSense 72 TCCGCTGCCGGGTTTTAACCCGACAGCAGTGACCTGTTTGAGCGAGAATTATAGTGAACCTCTTCGAGGGAC rpoB Sense Primer 20 ACGGCAACCATCAGATGGAG rpoB Antisense Primer 21 CGGTTTTAGTCTGCGCTTTCA rpoC-EGFP-Sense 87 GCCAGCCTGGCAGAACTGCTGAACGCAGGTCTGGGCGGTTCTGATAACGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpoC-EGFP-AntiSense 72 TAAAAAAACCCGCCGAAGCGGGTTTTTACGTTATTTGCGGATTAACGATTATAGTGAACCTCTTCGAGGGAC rpoC Sense Primer 19 CCGCAGGTGACTGCAGAAG rpoC Antisense Primer 24 ACAAATGCTCTTTCCCTAAACTCC rpoE-EGFP-Sense 87 TTCCGAGCGAGGGAAGCTATTGATAACAAAGTTCAACCGCTTATCAGGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpoE-EGFP-AntiSense 72 GTTGTTCTTTCTGCATGCCTAATACCCTTATCCAGTATCCCGCTATCGTCATAGTGAACCTCTTCGAGGGAC rpoE Sense Primer 20 TTGTCCGGTAGGTACGGTGC rpoE Antisense Primer 24 TTCGTTAAGCAGCTCACTATCCAG rpoH-EGFP-Sense 87 CGCCAGCTGGAAAAGAACGCGATGAAAAAATTGCGTGCTGCCATTGAAGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpoH-EGFP-AntiSense 72 ACAAAAACCCCGGACTCTCATCCAGGGTTCTCTGCTTAATAGCGGAAATTATAGTGAACCTCTTCGAGGGAC rpoH Sense Primer 21 AAGTCCACGTTGCAGGAACTG rpoH Antisense Primer 25 GAAATTGATTATTACAGAGGCCCAA rpoZ-EGFP-Sense 87 CAGGAAGCCGCTGAATTACAAGCCGTTACCGCTATTGCTGAAGGTCGTCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 214 rpoZ-EGFP-AntiSense 72 CAGTTGATTCAGGCTTTCAAACAGATACAAGGGCGACCCGCTTTGTGATTATAGTGAACCTCTTCGAGGGAC rpoZ Sense Primer 20 AACCAGATCCTCGACGTTCG rpoZ Antisense Primer 20 GGTCTTCCGGCAGGTAGGTT rpsA-EGFP-Sense 87 AACTTCTCCAACAACGCAATGGCTGAAGCTTTCAAAGCAGCTAAAGGCGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsA-EGFP-AntiSense 72 AAGTAAACTCAACAAACTTCGGAATAAAAATCCCGAAGAGTCAGAGAATTATAGTGAACCTCTTCGAGGGAC rpsA Sense Primer 23 GATGCAATCGCAACTGTTAACAA rpsA Antisense Primer 21 CAGGACGAAACCTGCAATCTG rpsB-EGFP-Sense 87 CGTTCTCAGGATCTGGCTTCCCAGGCGGAAGAAAGCTTCGTAGAAGCTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsB-EGFP-AntiSense 72 GCCGCCTTTCTGCAACTCGAACTATTTTGGGGGAGTTATCAAGCCTTATTATAGTGAACCTCTTCGAGGGAC rpsB Sense Primer 20 TTGCTGCAACCGTACGTGAA rpsB Antisense Primer 22 GCTCATCCCGGTCACTTACTGA rpsC-EGFP-Sense 87 CCGGAAAAACCGGCTGCTCAGCCTAAAAAGCAGCAGCGTAAAGGCCGTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsC-EGFP-AntiSense 72 CATTTTACGGAATTTTGTACGCTTTGGTTGTAACATCAGCGACGCTCCTTATAGTGAACCTCTTCGAGGGAC rpsC Sense Primer 21 GGTGGTATGGCTGCTGTTGAA rpsC Antisense Primer 20 CTGCCGAAGCTAACATCCGT rpsD-EGFP-Sense 87 GATCTGTCTGCGGACATTAACGAACACCTGATCGTCGAGCTTTACTCCAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsD-EGFP-AntiSense 72 AACTCTGTCACAGAACCCTGCATTGTGTCCTCTCTTTGGTACTAAGCTTTATAGTGAACCTCTTCGAGGGAC rpsD Sense Primer 20 ACGTTTAAGCGTAAGCCGGA rpsD Antisense Primer 22 ACTTGCTCGATATCAACCAGGC rpsE-EGFP-Sense 87 GAAATGGTCGCTGCCAAGCGTGGTAAATCCGTTGAAGAAATTCTGGGGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsE-EGFP-AntiSense 72 ACCGATTGCACTGCGGGTTTGAGTAATTTTAATAGTCTTTGCCATGGTTTATAGTGAACCTCTTCGAGGGAC rpsE Sense Primer 21 GCAACTATTGATGGCCTGGAA rpsE Antisense Primer 20 AGCAGCGTTGCCTTGTGTTT rpsF-EGFP-Sense 87 GATTTCGCAAACGAAACCGCTGATGATGCTGAAGCTGGGGATTCTGAAGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsF-EGFP-AntiSense 72 AGCCCTGCACACGGTGCCGGACAACACCAGACGGTTGGTCATCAGAAATTATAGTGAACCTCTTCGAGGGAC rpsF Sense Primer 22 ATGGTTAAAGCGAAAGACGAGC rpsF Antisense Primer 21 CCTGATGGACTGACCTTTCGA rpsG-EGFP-Sense 87 AGTCACCAGGCGGGCGCTTCCAGTAAGCAGCCCGCTTTGGGCTACTTAAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 215 rpsG-EGFP-AntiSense 72 GATGGGTGTTGTACGAGCCATTTGTTTCCTCGTTTATCTTTTAGGCGTTCATAGTGAACCTCTTCGAGGGAC rpsG Sense Primer 22 TCGCACACTACCGTTGGTTATC rpsG Antisense Primer 22 TGATACCGATGTTACGGTAGCG rpsH-EGFP-Sense 87 CGTGCAGCGCGCCAGGCTGGTCTTGGTGGCGAAATTATCTGCTACGTAGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsH-EGFP-AntiSense 72 AGGAACAACGACCGGTGCTTTAGCAACACGAGACATTTTTTCCTCCGATTATAGTGAACCTCTTCGAGGGAC rpsH Sense Primer 22 GATGAGCTGCCGAAAGTTATGG rpsH Antisense Primer 24 CTGACCGTTGATTTTTACGTCAAC rpsI-EGFP-Sense 87 AAGAAAGTCGGTCTGCGTAAAGCACGTCGTCGTCCGCAGTTCTCCAAACGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsI-EGFP-AntiSense 72 CCGAAGCGGGTTTTTTCGAAAATTGTTTTCTGCCGGAGCAGAAGCCAATTATAGTGAACCTCTTCGAGGGAC rpsI Sense Primer 21 TACTCGTGACGCTCGTCAGGT rpsI Antisense Primer 21 GCAGCAAAAAGCAACTTTCCA rpsJ-EGFP-Sense 87 CTGATGCGTCTGGATCTGGCTGCCGGTGTAGACGTGCAGATCAGCCTGGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsJ-EGFP-AntiSense 72 CGACTAAACCAATCATTGTTTCAACCTCTCAATCGCTCAATGACCTGATTATAGTGAACCTCTTCGAGGGAC rpsJ Sense Primer 20 TGGTTGACATCGTTGAGCCA rpsJ Antisense Primer 24 GTGAAGATACGGGTCATACCCACT rpsK-EGFP-Sense 87 GTGACTCCGATCCCTCATAACGGTTGTCGTCCGCCGAAAAAACGTCGCGTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsK-EGFP-AntiSense 72 CAAATATCTTGCCATTTTCTTTCTCCAACAAACCTGGAAAACGAGGCGTTATAGTGAACCTCTTCGAGGGAC rpsK Sense Primer 23 CAGGTTTCCGCATCACTAACATT rpsK Antisense Primer 20 TAAGTCGGTGCCCTCACGAC rpsL-EGFP-Sense 87 AAAGACCGTAAGCAGGCTCGTTCCAAGTATGGCGTGAAGCGTCCTAAGGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsL-EGFP-AntiSense 72 AGTTTGACATTTAAGTTAAAACGTTTGGCCTTACTTAACGGAGAACCATTATAGTGAACCTCTTCGAGGGAC rpsL Sense Primer 22 GTGTTCGTTACCACACCGTACG rpsL Antisense Primer 24 TTCAGGATTGTCCAAAACTCTACG rpsM-EGFP-Sense 87 ACCAAGACCAACGCACGTACCCGTAAGGGTCCGCGCAAACCGATCAAGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsM-EGFP-AntiSense 72 CGTTTACGTGCACGAATTGGTGCCTTTGCCATTATTCAATCACCCCGATTATAGTGAACCTCTTCGAGGGAC rpsM Sense Primer 19 TTGCGTCATCGTCGTGGTC rpsM Antisense Primer 19 GCCACGCCGTCAGAGACTT rpsN-EGFP-Sense 87 CGTGAAGCCGCTATGCGCGGTGAAATCCCGGGTCTGAAAAAGGCTAGCTGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 216 rpsN-EGFP-AntiSense 72 ATCTTGCATGCTCATCTGTCTTTACTCCCGTGATTCAATTGGTGACAATTATAGTGAACCTCTTCGAGGGAC rpsN Sense Primer 19 GGTTTCCTGCGGAAGTTCG rpsN Antisense Primer 20 CGTTACGGATACGGGTCAGC rpsO-EGFP-Sense 87 AAAGACGTAGCACGTTACACCCAGCTCATCGAGCGCCTGGGTCTGCGTCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsO-EGFP-AntiSense 72 CTTGAAAAAAGGGGCCACTCAGGCCCCCTTTTCTGAAACTCGCAAGAATTATAGTGAACCTCTTCGAGGGAC rpsO Sense Primer 24 TAAACTGCTCGACTACCTGAAACG rpsO Antisense Primer 19 TTCCAGTGAATTGCTGCCG rpsP-EGFP-Sense 87 ACTATTTCTGATCGCGTTGCTGCGCTGATCAAAGAAGTAAACAAAGCAGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsP-EGFP-AntiSense 72 AGGTGCTTGCGCGGTGAGTTGTTTGCTCATCATGACCACCGTGACAGATTATAGTGAACCTCTTCGAGGGAC rpsP Sense Primer 23 CAATCGCTAGCGAAAAAGAAGAA rpsP Antisense Primer 22 CCACCCACGAATACCGTAAGAC rpsQ-EGFP-Sense 87 TCCAAGACTAAATCCTGGACGCTGGTTCGCGTTGTAGAGAAAGCGGTTCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsQ-EGFP-AntiSense 72 AACGGCTCATTTCTGAGCCGTTTATTCGTATTGAGAGAGTGTACTGTATTATAGTGAACCTCTTCGAGGGAC rpsQ Sense Primer 21 GGTATCGGTGACGTGGTTGAA rpsQ Antisense Primer 20 CACCGCTTCAAGGATATGGG rpsR-EGFP-Sense 87 ATCAAACGCGCTCGCTACCTGTCCCTGCTGCCGTACACTGATCGCCATCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsR-EGFP-AntiSense 72 CTTGCATTACCTTATCCTCTCAAAGTCGTATTAATGGACCGTGACCGATTATAGTGAACCTCTTCGAGGGAC rpsR Sense Primer 20 AAATACCAGCGTCAGCTGGC rpsR Antisense Primer 22 TTACCTGATCACCCAGGCTACC rpsS-EGFP-Sense 87 CCGACTCGTACTTATCGCGGCCACGCTGCTGATAAAAAAGCGAAGAAGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsS-EGFP-AntiSense 72 AACGAGCATGGCGATGTTTAGCGATAGTTTCCATCTCTTCCTCCTACCTTATAGTGAACCTCTTCGAGGGAC rpsS Sense Primer 21 TGGTTGGTCACAAACTGGGTG rpsS Antisense Primer 21 AACAAGGCGAACCTTCTGAGC rpsT-EGFP-Sense 87 AAAGCTGCACGTCATAAGGCTAACCTGACTGCACAGATCAACAAACTGGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rpsT-EGFP-AntiSense 72 AAAACCCGCTTGCGCGGGCTTTTTCACAAAGCTTCAGCAAATTGGCGATTATAGTGAACCTCTTCGAGGGAC rpsT Sense Primer 22 CAGGCTGCTAAAGGTCTGATCC rpsT Antisense Primer 20 CATCACAAAAGCAGCAGGCA rpsU-EGFP-Sense 87 CGTCACGCGAAGAAACTGGCTCGCGAAAACGCACGCCGCACTCGTCTGTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 217 rpsU-EGFP-AntiSense 72 CCTTACAACTACAACTCGGTCTGATCGGAGAGCAACGCTCTCGGGGAATTATAGTGAACCTCTTCGAGGGAC rpsU Sense Primer 21 GCGCTAAAGCTTCTGCAGTGA rpsU Antisense Primer 19 TAAGCCGCGCATTCCTTTC rsmB-EGFP-Sense 87 CCTGGTGCCGAAGAGGGCGACGGCTTCTTTTACGCTAAGCTAATCAAAAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG rsmB-EGFP-AntiSense 72 GCCGGCACCCAGAATGATAATTTTCATCAGTCGCGACCCGTTATCTCATCATAGTGAACCTCTTCGAGGGAC rsmB Sense Primer 20 GTACCGCTGATGCCGAACTT rsmB Antisense Primer 20 TTCTCGCCAACCAGGTTTTC ruvA-EGFP-Sense 87 CCTGACGCCAGCAGTGAAACTTTAATTCGCGAAGCCCTACGCGCCGCGTTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ruvA-EGFP-AntiSense 72 AAGTGGTACCGGCAGAAATCAGACGGTCTGCTTCAATCATCCTTTACCTCATAGTGAACCTCTTCGAGGGAC ruvA Sense Primer 20 AAACCACAAGAAGCAAGCCG ruvA Antisense Primer 21 GATCTGCTACATCTTCCGGCA ruvB-EGFP-Sense 87 GCGACGACGCGGGCGTGGAATCACTTTGGCATAACGCCGCCAGAAATGCCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ruvB-EGFP-AntiSense 72 ATCAGGCATATTGCCAGTGCCGGATGCGGCGCGAGCGACCAATCCGACTTATAGTGAACCTCTTCGAGGGAC ruvB Sense Primer 23 AACCTTATTTGATTCAGCAAGGC ruvB Antisense Primer 22 TAGTCCTGGTAAGACGCGAACA ruvC-EGFP-Sense 87 GCGATGCAGATGAGCGAATCGCGGCTGAACCTGGCGAGAGGGCGACTGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ruvC-EGFP-AntiSense 72 CGGAGGGAGTGGAAACGCCTCAGCCGGAACTGACCGAGGCGGTATAACTTATAGTGAACCTCTTCGAGGGAC ruvC Sense Primer 20 CCCACTGCCACGTTAGTCAG ruvC Antisense Primer 20 ATCCGGGTTTGGTGTTGATT sbcB-EGFP-Sense 87 GAGAAAGTGGCGCTGTTAAAAGCACTTTGGCAGTACGCGGAAGAGATTGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sbcB-EGFP-AntiSense 72 GCCTGACTCAACATTGTCCTCCGCCGTACCAGCGGCGGAGGCTTCAAATTATAGTGAACCTCTTCGAGGGAC sbcB Sense Primer 24 TGCAGATGCTGGTACAACAATATG sbcB Antisense Primer 23 ATCACCGATTATCAGCAGATTGG sdhA-EGFP-Sense 87 AACATGGAACCGAAACTGCGCCCGGCATTCCCGCCGAAGATTCGTACTTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sdhA-EGFP-AntiSense 72 GTTATAGCGATAAATTGAAAACTCGAGTCTCATTTTCCTGTCTCCGCATTATAGTGAACCTCTTCGAGGGAC sdhA Sense Primer 18 GTCGGAATCCATGACGCG sdhA Antisense Primer 20 CATACGCGGAGCATCATCAA sdhB-EGFP-Sense 87 CCGACGCGCGCCATCGGCCATATCAAGTCGATGTTGTTGCAACGTAATGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 218 sdhB-EGFP-AntiSense 72 GCACTGGTTGCCTGATGCGACGCTTGCGCGTCTTATCAGGCCTACGGTTTATAGTGAACCTCTTCGAGGGAC sdhB Sense Primer 24 TCATGAACTGCGTCAGTGTATGTC sdhB Antisense Primer 19 TGTAGGCCGGATAAGGCGT secA-EGFP-Sense 87 TGCCCGTGCGGTTCTGGTAAAAAATACAAGCAGTGCCATGGCCGCCTGCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG secA-EGFP-AntiSense 72 TATAAAAAAGGCGCAGAATCCTGCGCCTTTTACTTCAACAGTTAGCTTTTATAGTGAACCTCTTCGAGGGAC secA Sense Primer 20 AAACCGGAGAGCGCAAAGTA secA Antisense Primer 24 CAATTTGCAGCTTTTTCATTGTCT secB-EGFP-Sense 87 AACTATTTGCAGCAGCAGGCTGGCGAAGGTACTGAAGAACATCAGGATGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG secB-EGFP-AntiSense 72 CGTACGAGCCGGCACCGATCACAGTCATTGAAGCATTACGTTGGTTCATCATAGTGAACCTCTTCGAGGGAC secB Sense Primer 21 ACATTCCCGCAACTGAACCTT secB Antisense Primer 18 GGCCATTTCTTGCCAGGG secD-EGFP-Sense 87 GCCATCGTAAACCTGCTATATGGCGGCAAGCGCGTCAAGAAGCTGTCAATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG secD-EGFP-AntiSense 72 CCGTGGTTTAGTTGTTCAACAGTATATTCCTGTGCCACATCGCACTCCTCATAGTGAACCTCTTCGAGGGAC secD Sense Primer 20 TGTGGCGACGTCGATGTTTA secD Antisense Primer 23 CCCAGCGCATAAAGTCATAGACT secE-EGFP-Sense 87 CTGGATGGTATTCTGGTTCGCCTGGTATCCTTTATCACTGGCCTGAGGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG secE-EGFP-AntiSense 72 GAAAACGCCTGAACGACGTACCAGCGCTTTTTAGGAGCTTCAGACATCTCATAGTGAACCTCTTCGAGGGAC secE Sense Primer 23 ACCGCAGTAATGTCACTGATCCT secE Antisense Primer 18 TGCTACGCGGCCTTCAAA secF-EGFP-Sense 87 TTGCAGCAGAAAGTGGAAAAAGAAGGGGCGGATCAGCCGTCAATTCTGCCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG secF-EGFP-AntiSense 72 AAAATCCCGATCTTCTGACCGGGATTTTCAACATCAACGGGAACTTGATTATAGTGAACCTCTTCGAGGGAC secF Sense Primer 19 TGGGTATGAAGCGCGAACA secF Antisense Primer 22 GGGATTTTCGCAATCTCCATAC secG-EGFP-Sense 87 GAACAAACTCAGCCAGCTGCTCCGGCTAAGCCGACCAGCGATATCCCGAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG secG-EGFP-AntiSense 72 AAGGTAGCGTGTCTACCAATTCCACCACCTCGGCACGGATACTACTTTTTATAGTGAACCTCTTCGAGGGAC secG Sense Primer 22 AGCGAATGGGAAAATCTGAGTG secG Antisense Primer 22 CTTGAACCCGTAAGCCCTATTG secY-EGFP-Sense 87 AGTCAGTATGAGTCTGCATTGAAGAAGGCGAACCTGAAAGGCTACGGCCGACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 219 secY-EGFP-AntiSense 72 GAAGCACGAACTTTCATTTTTACTCTCCGTAACTTCTCGGGCGACCAATTATAGTGAACCTCTTCGAGGGAC secY Sense Primer 25 ATTATGGACTTTATGGCTCAAGTGC secY Antisense Primer 22 CGATTTTGCAGTTACGGCATAA serA-EGFP-Sense 87 CTGCAGGCAATGAAAGCTATTCCGGGTACCATTCGCGCCCGTCTGCTGTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG serA-EGFP-AntiSense 72 AAAACGGGCAAGTCAGTGACCTGCCCGTTGATTTTCAGAGAAGGGGAATTATAGTGAACCTCTTCGAGGGAC serA Sense Primer 20 TTGAAGCCGACGAAGACGTT serA Antisense Primer 19 GCAACGGTGTGGAGAAGGG serC-EGFP-Sense 87 GGCGTTAAAGCGCTGACAGACTTCATGGTTGAGTTCGAACGCCGTCACGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG serC-EGFP-AntiSense 72 AATAAAAACCCCACAGGCTGGCTGTGGGGATTAAGCAAAATTTCGGCATTATAGTGAACCTCTTCGAGGGAC serC Sense Primer 21 ATGCACTGAAAGGTCACCGTG serC Antisense Primer 21 GAGCGATGGGTTGTAACGTCA serS-EGFP-Sense 87 GAAGTACCAGAAGTTCTGCGTCCGTATATGAACGGACTGGAATATATTGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG serS-EGFP-AntiSense 72 ACAAAAAAAGCGCCCGCAGGCGCTTTTTAGATTCAGAAAAATTGGGTATTATAGTGAACCTCTTCGAGGGAC serS Sense Primer 21 CTATCAGCAGGCTGATGGTCG serS Antisense Primer 27 GTTCGCTATATAGGCTTGTATACATCG slyD-EGFP-Sense 87 GGTGGCGAAGGCTGCTGTGGCGGTAAAGGCAACGGCGGTTGCGGTTGCCACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG slyD-EGFP-AntiSense 72 GGCGTAAAAAAAGCGGGGATTCCCCGCTTTTTTGTCACTTTTTCGGTATTATAGTGAACCTCTTCGAGGGAC slyD Sense Primer 23 AAGAAGAACTGGCTCATGGTCAC slyD Antisense Primer 22 GCTGACCGAGAAGTTAAAAGCC slyX-EGFP-Sense 87 CAGCCGTCGAACATCGCGTCGCAGGCTGAAGAAACGCCACCGCCACATTATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG slyX-EGFP-AntiSense 72 TACCGAAAAAGTGACAAAAAAGCGGGGAATCCCCGCTTTTTTTACGCCTCATAGTGAACCTCTTCGAGGGAC slyX Sense Primer 20 CGATCATCTGCGTCTGCTGA slyX Antisense Primer 19 GTCATGAACACGGTGGCGA smF-EGFP-Sense 87 TATGTCCGATTGAGGAGGGCATGCCATGTTCGACGTACTAATGTATTTGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG smF-EGFP-AntiSense 72 AGTTTGTCTTGATCCACACGCAACTCAGCTTCTGTGTGAATATAGGTTTCATAGTGAACCTCTTCGAGGGAC smF Sense Primer 19 AGCAGGATGGATCGCAGCT smF Antisense Primer 20 CCTGCGTCGGTAAGATCCTG smpB-EGFP-Sense 87 GAGCGCGAATGGCAGGTGGATAAAGCACGTATCATGAAAAACGCCCACCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 220 smpB-EGFP-AntiSense 72 CGGAGAGGGAGGCGCGGTGAGGAACTGGTCAATAATTGGAGTGCAGGTTTATAGTGAACCTCTTCGAGGGAC smpB Sense Primer 21 CGCCAAAGGTAAGAAACAGCA smpB Antisense Primer 22 TTGTGTCGGATGTGATAGCCAA sohB-EGFP-Sense 87 GCCGATCGATTGTTGCTACGCTGGTGGCAGCGGGGTCAAAAGCCATTGATGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sohB-EGFP-AntiSense 72 TCTGGTTGATTAAAGCAAAACGCGAGGTCTTAGCCTCGCGTTTGTCTTTTATAGTGAACCTCTTCGAGGGAC sohB Sense Primer 20 CTCATTGACCGATTCACCGG sohB Antisense Primer 20 GGCCCATGTGCTTTCAGAAA spoT-EGFP-Sense 87 CGCAAAATCCGCGTGATGCCAGACGTGATTAAAGTCACCCGAAACCGAAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG spoT-EGFP-AntiSense 72 GGCGAGCATTTCGCAGATGCGTGCATAACGTGTTGGGTTCATAAAACATTATAGTGAACCTCTTCGAGGGAC spoT Sense Primer 21 AGCGCCTTTATTCGTCTGACC spoT Antisense Primer 20 TTTGTGGACCTGCTCCATGC ssB-EGFP-Sense 87 GCAGCGCCGTCTAACGAGCCGCCGATGGACTTTGATGATGACATTCCGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ssB-EGFP-AntiSense 72 TTTAATCATCCACCTTAAAACAATATAACCTATTGTTTTAATGACAAATCATAGTGAACCTCTTCGAGGGAC ssB Sense Primer 18 AGTCTCGCCCGCAGCAGT ssB Antisense Primer 19 AAGCCTCCATAACGGAGGC sspA-EGFP-Sense 87 TTCCTTGCTTCTTTAACTGAAGCAGAACGTGAAATGCGTCTGGGCCGGAGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sspA-EGFP-AntiSense 72 GCAGCAGATAGGGACGACGTGGTGTTAGCTGTGACAAATCCATACAGATTATAGTGAACCTCTTCGAGGGAC sspA Sense Primer 21 GAAAGGCTATATGACCCGCGT sspA Antisense Primer 24 TGGTTATCCAGCAACCACTCATAG sspB-EGFP-Sense 87 GAACCTCCGCAGCCACCACGCGGTGGTCGACCGGCATTACGCGTTGTGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sspB-EGFP-AntiSense 72 TATTAAGTCATTAAAAAGACAAAACAGGCCGCCTGGGCCTGTTTTGTATTATAGTGAACCTCTTCGAGGGAC sspB Sense Primer 24 GATGATGACACTCATCCTGACGAT sspB Antisense Primer 22 AATATGATTGCCACAATCACGC sucA-EGFP-Sense 87 GTTCACCAGAAACAGCAACAAGATCTGGTTAATGACGCGCTGAACGTCGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sucA-EGFP-AntiSense 72 GCAGGTCAGGGACCAGAATATCTACGCTACTCATTGTGTATCCTTTATTTATAGTGAACCTCTTCGAGGGAC sucA Sense Primer 21 CCAGCATCATTTCCGTGAAGT sucA Antisense Primer 19 GTGGCATCGGCTACGGATT sucB-EGFP-Sense 87 GTAACGATCAAAGAGTTGCTGGAAGATCCGACGCGTCTGCTGCTGGACGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 221 sucB-EGFP-AntiSense 72 AGGCGATAATGCCTTATCCGGTCTACAGTGCAGGTGAAACTTAAACTACTATAGTGAACCTCTTCGAGGGAC sucB Sense Primer 20 CGCTGTCCTACGATCACCGT sucB Antisense Primer 20 CGTCGCATCAGGCTTCAATT sucC-EGFP-Sense 87 AAAGGTCTGACGGATGCAGCTCAGCAGGTTGTTGCCGCAGTGGAGGGGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sucC-EGFP-AntiSense 72 TAAAGCCCTGGCAGATAACCTTGGTGTTTTTATCGATTAAAATGGACATTATAGTGAACCTCTTCGAGGGAC sucC Sense Primer 23 CTGACAGCGGCCTGAATATTATT sucC Antisense Primer 21 TGGCCTGTTCTGAGTGGAAAG sucD-EGFP-Sense 87 ACCGTTCGCAGCCTGGCGGATATCGGTGAAGCACTGAAAACTGTTCTGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG sucD-EGFP-AntiSense 72 CCATTCCTGTAGAGGGAAGCGGCGAGGGCTATTTCTTATTACAGATATTTATAGTGAACCTCTTCGAGGGAC sucD Sense Primer 22 GAGAAATTCGCTGCTCTGGAAG sucD Antisense Primer 20 CCAACCATGTCGAAACCGAC suhB-EGFP-Sense 87 AAAGCCATGCTGGCGAACATGCGTGACGAGTTAAGCGACGCTCTGAAGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG suhB-EGFP-AntiSense 72 AGCGCCTGAAAGGCGAGGGCGGGTGAGTGATATCACCCGCCTGAGTCATTATAGTGAACCTCTTCGAGGGAC suhB Sense Primer 21 TGACGAGTTAAGCGACGCTCT suhB Antisense Primer 22 GAAAGATGGTGGGAGAGTGACG surE-EGFP-Sense 87 CAAGATGTGGTTTCAGACTGGTTAAACAGCGTGGGAGTTGGCACGCAATGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG surE-EGFP-AntiSense 72 GAATACCTTGCGCACGTAATTGATCCAGAAGTGCTTGTACGCGTCTGCTTATAGTGAACCTCTTCGAGGGAC surE Sense Primer 21 CCGCTGCATGTGGATTTAACT surE Antisense Primer 21 AAGTGCATTCAGCACCTGCTC taG-EGFP-Sense 87 CTGGTGAATGATCATGTGGTTGGCTGCTGTTGCTATCCGGGAAATAAACCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG taG-EGFP-AntiSense 72 GCCACAGTTCGAGGATCGCGGGGAGTTCTGAACGTTGCGCTTCCCGAATCATAGTGAACCTCTTCGAGGGAC taG Sense Primer 25 ATCTGTTACTCCTTTATGCAGGCAT taG Antisense Primer 21 CAGTCACGCCAGTAATTCGCT talB-EGFP-Sense 87 AAGTTTGCTATTGACCAGGAAAAACTGGAAAAAATGATCGGCGATCTGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG talB-EGFP-AntiSense 72 GCTTCAGAAGAGGTAGCGTGACCGACTTCCCGGTCACGCTAAGAATGATTATAGTGAACCTCTTCGAGGGAC talB Sense Primer 22 CGCGTATCACTGAGTCCGAGTT talB Antisense Primer 21 CACTGCGAAGGGAGTGACAGA tatC-EGFP-Sense 87 AATCGGGAAGAGGAAAACGACGCTGAAGCAGAAAGCGAAAAAACTGAAGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 222 tatC-EGFP-AntiSense 72 CAAACATCCTGTACTCCATATGACAACCGCCCTGACGGGCGGTTGAATTTATAGTGAACCTCTTCGAGGGAC tatC Sense Primer 24 TGTCTTCTTCTCACGCTTTTACGT tatC Antisense Primer 23 ATTGCGAACTGGTCAAATTAACG tgT-EGFP-Sense 87 ACTGATTTTTACCAGCGTCAGGGGCGAGAAGTACCACCTTTGAACGTTGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tgT-EGFP-AntiSense 72 ACCGCATCAGAAATAAAAAAGCTCATTAAATTTCCCTCATTATTAATATTATAGTGAACCTCTTCGAGGGAC tgT Sense Primer 21 ACGCAAGGCTATTGAAGAGGG tgT Antisense Primer 23 AGCATCAAAATCAAAGACATCGG thiL-EGFP-Sense 87 GGCGAACCTGTTACATTAGACTGGAAAGGATATGACCATTTTGCCACGCCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG thiL-EGFP-AntiSense 72 AGTAGATGCCACGGATTACTCATCTTCAGGCGACTTTTCGCGACATCTTTATAGTGAACCTCTTCGAGGGAC thiL Sense Primer 23 TTTACCTGTATCGGGCAAATGAC thiL Antisense Primer 22 TTAATCCACTTCCGAATCCGAC thrS-EGFP-Sense 87 GAGAAGCTGCAACAAGAGATTCGCAGCCGCAGTCTTAAACAATTGGAGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG thrS-EGFP-AntiSense 72 GATACGGTTAGGGCGCGCCGTTTGAACTCGTTTTCCGCCTTTAATACCTTATAGTGAACCTCTTCGAGGGAC thrS Sense Primer 21 CGTGGTAAAGACCTGGGAAGC thrS Antisense Primer 22 ACCTGTTAAGCGAACTTCCTGG thyA-EGFP-Sense 87 GAGATTGAAGGCTACGATCCGCATCCGGGCATTAAAGCGCCGGTGGCTATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG thyA-EGFP-AntiSense 72 AAAAAAACCGACGCACACTGGCGTCGGCTCTGGCAGGATGTTTCGTAATTATAGTGAACCTCTTCGAGGGAC thyA Sense Primer 23 TTCGACTACCGTTTCGAAGACTT thyA Antisense Primer 22 AGGCGTCTCGAAGAATTTAACG tiG-EGFP-Sense 87 AAAGTGACTGAAAAAGAAACCACTTTCAACGAGCTGATGAACCAGCAGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tiG-EGFP-AntiSense 72 TGGCGGTGACGGGCCTTTGTGCGAATTTAGCGCGTTATGCTGCGTAAATTATAGTGAACCTCTTCGAGGGAC tiG Sense Primer 23 ACAGGCTGTTGAAGCTGTACTGG tiG Antisense Primer 19 GCTTTTCGCACGGTTCCAT tktA-EGFP-Sense 87 GAGTTCGGCTTCACTGTTGATAACGTTGTTGCGAAAGCAAAAGAACTGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tktA-EGFP-AntiSense 72 AGGTAATAAAAAAGGTCGCCGAAGCGACCTTTTTACCCGAAATGCTAATTATAGTGAACCTCTTCGAGGGAC tktA Sense Primer 20 CCACCTTCGGTGAATCTGCT tktA Antisense Primer 21 CTTGTCCGCAAACGGACATAT tldD-EGFP-Sense 87 GTGGGCCAGCCAACGTTGAAAGTCGATAACCTGACTGTTGGCGGTACTGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 223 tldD-EGFP-AntiSense 72 TTCGTGCACGTAGAAAGATTAATTATCCTTCTGAAAATAGTGAAATTATTATAGTGAACCTCTTCGAGGGAC tldD Sense Primer 21 GTGGGTGTCTGCGGTAAAGAA tldD Antisense Primer 22 ACGTTGTACAAACCTGTGCCAA tmK-EGFP-Sense 87 ATGGATGCAATCCGCACTACCGTGACCCACTGGGTGAAGGAGTTGGACGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tmK-EGFP-AntiSense 72 AGCTGGCTACCAGTTTTTCGAAATCAGGTCGTAACCATGGATACCATCTCATAGTGAACCTCTTCGAGGGAC tmK Sense Primer 22 ATTCATACCATTGATGCCACCC tmK Antisense Primer 22 AGTAGCGCATGGTGACCTCTTC tolB-EGFP-Sense 87 CTTCCGGCAACTGATGGACAGGTCAAATTCCCTGCCTGGTCGCCGTATCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tolB-EGFP-AntiSense 72 TTGTTCAGTTGCATTTCAATGATTCCTTTACTATTCAATTAATTATTATCATAGTGAACCTCTTCGAGGGAC tolB Sense Primer 22 ATTTGGTTTCTACAGATGGGCG tolB Antisense Primer 21 GAGCAATCATCAGCCCTTTCA tolC-EGFP-Sense 87 CAAACATCCGCACGCACTACCACCAGTAACGGTCATAACCCTTTCCGTAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tolC-EGFP-AntiSense 72 CTTTACGTTGCCTTACGTTCAGACGGGGCCGAAGCCCCGTCGTCGTCATCATAGTGAACCTCTTCGAGGGAC tolC Sense Primer 22 CTGATGGTTATGCGCCTGATAG tolC Antisense Primer 20 GGGAAGAATGCGGCAGATAA tolQ-EGFP-Sense 87 ACCGCGATTCTGCACCGCCAGGCGTTTACCGTTAGCGAGAGCAACAAGGGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tolQ-EGFP-AntiSense 72 TTCGGACTTGAGATCGCGACGACCTCGTCCACGCGCTCTGGCCATGGCTTATAGTGAACCTCTTCGAGGGAC tolQ Sense Primer 27 GAACTGAATTACGACAACTTTATGGAA tolQ Antisense Primer 21 CGTCCAGCAACGGTACAATGT tolR-EGFP-Sense 87 TTGTTACATAGTGCGGGTGTGAAATCGGTTGGTTTAATGACGCAGCCTATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tolR-EGFP-AntiSense 72 CTGTTCGCCTGTTACCCGCTCTCTTTCAAGCAAGGGAAACGCAGATGTTTATAGTGAACCTCTTCGAGGGAC tolR Sense Primer 22 GCAAAAGATGTGCCTTACGATG tolR Antisense Primer 22 TTTGACACTCTCGGTTTCCAAA topA-EGFP-Sense 87 ACTGGCTGGTCAGCATTTTATGTTGATGGCAAATGGGTTGAAGGAAAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG topA-EGFP-AntiSense 72 GGCCGCTTTCGCGACCCTTTGTTTATAAAAACCTGACAGAATTAAAGGTTATAGTGAACCTCTTCGAGGGAC topA Sense Primer 21 CTTCGGAAAAAGACGGAAAGG topA Antisense Primer 21 TCGTTTTTTCGTCACACCTGG tpiA-EGFP-Sense 87 GCTGACGCCTTCGCAGTAATCGTTAAAGCTGCAGAAGCGGCTAAACAGGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 224 tpiA-EGFP-AntiSense 72 GAGTTAAGGAAAGTAAGTGCCGGATATGAAATCCGGCACCTGTCAGACTTATAGTGAACCTCTTCGAGGGAC tpiA Sense Primer 20 GTTTGCTCAGCCGGATATCG tpiA Antisense Primer 23 AGTGTGAGATTTTGCGTTAAGGC trmD-EGFP-Sense 87 GAGTTCAAAACGGAACACGCACAACAGCAACATAAACATGATGGGATGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trmD-EGFP-AntiSense 72 TGCTCATAATTTAATCTCTTATCCTGGGTAAACTGATATCTCGGGGGCTTATAGTGAACCTCTTCGAGGGAC trmD Sense Primer 21 CTGAAGAGCAAGCAAGGTTGC trmD Antisense Primer 23 GTCCTGCTTCATCTGCTCTTGTT trmU-EGFP-Sense 87 GAAGTGTGCCTCGGTGGCGGTATTATTGAGCAGCGTCTGCCGCTGCCGGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trmU-EGFP-AntiSense 72 AATTCTTTGCCACGTTCACTGCTTCCTTGTTTTAAGTAAAGATAATAATCATAGTGAACCTCTTCGAGGGAC trmU Sense Primer 23 CCAGTCTGCCGTCTTCTATAACG trmU Antisense Primer 20 AGGGCGAGGGTGATGTCATA trpA-EGFP-Sense 87 CTGGCGGCACTGAAAGTTTTTGTACAACCGATGAAAGCGGCGACGCGCAGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trpA-EGFP-AntiSense 72 TCATTAAAGAAAGTTAAAATGCCGCCAGCGGAACTGGCGGCTGTGGGATTATAGTGAACCTCTTCGAGGGAC trpA Sense Primer 24 TCGAGCAACATATTAATGAGCCAG trpA Antisense Primer 19 CGGGGTAAGCGAAACGGTA trpB-EGFP-Sense 87 GATAAAGACATCTTCACCGTTCACGATATTTTGAAAGCACGAGGGGAAATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trpB-EGFP-AntiSense 72 CTTCTTTGCGCTCCTTCAACTGGGCAAACAGAGATTCGTAGCGTTCCATCATAGTGAACCTCTTCGAGGGAC trpB Sense Primer 24 AGCAGCTACTGGTGGTTAACCTTT trpB Antisense Primer 20 AGCGTGACGAAAGGAACGAA trpE-EGFP-Sense 87 CGCGCTGTACTGCGCGCTATTGCCACCGCGCATCATGCACAGGAGACTTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trpE-EGFP-AntiSense 72 GGTTGTACGTAAAAGAGTCGATATTATCGAGCAGCAGAATGTCAGCCATCATAGTGAACCTCTTCGAGGGAC trpE Sense Primer 19 AAGCCGACGAAACCCGTAA trpE Antisense Primer 21 ATCACCACGTTATGCCCATTG trpS-EGFP-Sense 87 TCCCGTACGCTAAAAGCGGTGTACGAAGCGATTGGTTTTGTGGCGAAGCCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trpS-EGFP-AntiSense 72 CACGCCGCATCCGGCATGAACAAAGCGCAATTTGCCAGCAATAGTGAATTATAGTGAACCTCTTCGAGGGAC trpS Sense Primer 22 TCCTGCAACAGGTGATGAAAGA trpS Antisense Primer 21 TGCATTACTTTGTAGGCCGGA truA-EGFP-Sense 87 GACCGGTATGATCTTCCAAAACCGCCAATGGGCCCGCTATTTCTGGCGGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 225 truA-EGFP-AntiSense 72 CTGTAACTACGTTGGCTACAGCCTGAAATGTTCCGAGCATTATTCTCGTTATAGTGAACCTCTTCGAGGGAC truA Sense Primer 23 AGAGCTGGATAGCAGAGTTGCTG truA Antisense Primer 23 GCCTATGCCTTGTACTCGTCATC trxA-EGFP-Sense 87 GGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trxA-EGFP-AntiSense 72 GCCGGGCGTCCAGTTTTTAGCGACGGGGCACCCGAACATGAAATTCCCTTATAGTGAACCTCTTCGAGGGAC trxA Sense Primer 23 CTGCTGTTCAAAAACGGTGAAGT trxA Antisense Primer 21 GATAAAACGGGAAGCGTTGGA trxB-EGFP-Sense 87 ATGGCAGCACTTGATGCGGAACGCTACCTCGATGGTTTAGCTGACGCAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG trxB-EGFP-AntiSense 72 ATAGTCGCATGGTGTCGCCTTCTTTACTTTTGTTACTGATTTGTAAAATTATAGTGAACCTCTTCGAGGGAC trxB Sense Primer 20 GGCGACGTGATGGATCACAT trxB Antisense Primer 22 TTGCAGAGCAATGTTACAACGG tsF-EGFP-Sense 87 AAAGTTGAGACTGACTTTGCAGCAGAAGTTGCTGCGATGTCCAAGCAGTCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tsF-EGFP-AntiSense 72 AAGATGGGCACAAAAAGAAGCCGCCCTCAGGCGGCTCCTTTTTGATAATTATAGTGAACCTCTTCGAGGGAC tsF Sense Primer 21 CGCTGAAGTGACTGGCTTCAT tsF Antisense Primer 21 CCCCACAAGGGTTAGCTGAAT tufB-EGFP-Sense 87 CGTGAAGGCGGCCGTACCGTTGGCGCGGGCGTTGTAGCAAAAGTTCTGAGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tufB-EGFP-AntiSense 72 ATCAAATGATGCCCTTTTAGTGCGCATTGCGTCAAATGTTATCGGCAATTATAGTGAACCTCTTCGAGGGAC tufB Sense Primer 22 AAGGCGTAGAGATGGTAATGCC tufB Antisense Primer 20 TTCTGGTACGAAAGCGTGCA typA-EGFP-Sense 87 GACGAACTGGTAGAAGTGACTCCGACCTCTATCCGTATTCGTAAACGTCACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG typA-EGFP-AntiSense 72 ATTAATCGTCTTTCGGTGCGCGGTTGGCGCGGCGACGATCGTTTTCCGTCATAGTGAACCTCTTCGAGGGAC typA Sense Primer 23 TGGAACAAGCTCTGGAGTTCATC typA Antisense Primer 21 CGCTGGCAGGTTTTTTATGAC tyrS-EGFP-Sense 87 TTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGCTGGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG tyrS-EGFP-AntiSense 72 TAAAAACCAGGGGGAGTGATTTCTCACTCCCCCTTTCCACTTAATGCATTATAGTGAACCTCTTCGAGGGAC tyrS Sense Primer 23 GAAGAAGATCGTCTGTTTGGTCG tyrS Antisense Primer 22 CAACGTGAGACTGGATAGCGAG ubiA-EGFP-Sense 87 TATGTTGGTCTGGTACTATTTTTAGGGCTGGCAATGAGTTACTGGCATTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 226 ubiA-EGFP-AntiSense 72 CAGGCAACCCAGAAGAAAGCCGGATGATCATCCGGCTTTTTTACATCATCATAGTGAACCTCTTCGAGGGAC ubiA Sense Primer 19 TGGCGCGCTGTTTGTTTAT ubiA Antisense Primer 21 GTTGTGTAGGCCTGAGAAGCG ubiE-EGFP-Sense 87 TACTACAATCTGACGGCAGGGGTTGTGGCGCTGCATCGTGGTTATAAGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ubiE-EGFP-AntiSense 72 TCAATTCCTGCCGTCACTAAAGGTTTAAAAGGCATTTCCGGTCTCCTGTCATAGTGAACCTCTTCGAGGGAC ubiE Sense Primer 20 AAAGCCATGATGCAGGATGC ubiE Antisense Primer 22 CGATACAGGAAGGTGTTGAGCA ubiG-EGFP-Sense 87 AAACTCGGCCCCGGCGTGGATGTGAACTATATGCTGCACACGCAGAATAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ubiG-EGFP-AntiSense 72 AGACGCGGCAAGCGTCGCATCAGGCATTGGCGCGGCCAAACATCAACCTCATAGTGAACCTCTTCGAGGGAC ubiG Sense Primer 23 CATATCACTGGGCTGCATTACAA ubiG Antisense Primer 20 GCCTACGGGGAGCATTTGTA ubiH-EGFP-Sense 87 ACCCCGGCACGCGATGTGCTGGCGCAGCGCACCCTCGGTTGGGTGGCGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ubiH-EGFP-AntiSense 72 ACAATGGCTACATCAACACTTTGCATTGTTTATTCCTTAAAACCGCCTTCATAGTGAACCTCTTCGAGGGAC ubiH Sense Primer 20 AACATCGGGCTGATGACGAT ubiH Antisense Primer 20 CCCTGTAAGCCACAGGCAAC unG-EGFP-Sense 87 CGTGGCGAGACGCCGATTGACTGGATGCCAGTATTACCGGCAGAGAGTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG unG-EGFP-AntiSense 72 ATAAATCAGCCGGGTGGCAACTCTGCCATCCGGCATTTCCCCGCAAATTTATAGTGAACCTCTTCGAGGGAC unG Sense Primer 22 ATTTTGTGCTGGCAAATCAGTG unG Antisense Primer 22 TGAATGGTGGCGTCAGAATAAA upP-EGFP-Sense 87 TACATTATTCCGGGCCTCGGCGATGCCGGTGACAAAATCTTTGGTACGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG upP-EGFP-AntiSense 72 CTCAAAAAAAAGCCGACTCTTAAAGTCGGCTTTAATTATTTTTATTCTTTATAGTGAACCTCTTCGAGGGAC upP Sense Primer 22 ATTGATCAGGGACTGAACGAGC upP Antisense Primer 19 GATAGCACGGCGCGTCATA usG-EGFP-Sense 87 GCGCTGATGGCAGTAAAAATCGCCGAGAAACTGGTGCAGGAGTATCTGTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG usG-EGFP-AntiSense 72 CAATGCCCAGCGCAATTTTATAAACTGGCGGTTGTTGCTGGTCGGACATTATAGTGAACCTCTTCGAGGGAC usG Sense Primer 19 GTCGGTGGCCGATAACGTT usG Antisense Primer 23 CAGCCGTAATACTTACTGCCGTC uup Antisense Primer 23 CATGCCTGATAAACCCAGTTCAT 227 uup Sense Primer 23 AAAAAGTGCTTGCTGATATGGCT uup-EGFP-AntiSense 72 CAATTTTACCGTTGTCAGCTGTCTCTGTTTAAATCGACTATTTTGCGATCATAGTGAACCTCTTCGAGGGAC uup-EGFP-Sense 87 GAGCAAGCCTTTGAACGCTGGGAGTATCTTGAAGCGTTAAAAAATGGTGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG uvrA-EGFP-Sense 87 GTCGCGGAGTGCGAAGCATCACACACGGCACGCTTCCTTAAGCCGATGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG uvrA-EGFP-AntiSense 72 TGGTGCAACTCTGAAAGGAAAAGGCCGCTCAGAAAGCGGCCTTAACGATTATAGTGAACCTCTTCGAGGGAC uvrA Sense Primer 20 AGATCCTCGTCTCCGGTACG uvrA Antisense Primer 21 TGCCGGAAGAAAAACGTAAAT uvrB-EGFP-Sense 87 CAAATTCGTGACCAGTTGCATCAGCTGCGTGAGCTGTTTATCGCGGCATCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG uvrB-EGFP-AntiSense 72 GCGCATCAGGCTGTTTTCCGTTTGTCATCAGTCTTCTTCGCTATCCTGTTATAGTGAACCTCTTCGAGGGAC uvrB Sense Primer 19 GCGCAGAATCTGGAGTTCG uvrB Antisense Primer 23 GAAAATGTAGGCCTGATAAGCGT uvrC-EGFP-Sense 87 CCGGGTATTTCGCAAGGTCTGGCAGAAAAGATCTTCTGGTCGTTGAAACATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG uvrC-EGFP-AntiSense 72 GTAACTATCTGTTGTCAGTAAGATTACCCCTATGTTGCTACAGAGACATCATAGTGAACCTCTTCGAGGGAC uvrC Sense Primer 19 TTACGTAACGCCAGCGTCG uvrC Antisense Primer 28 ACGTAGGGATATTAAATTGCATAATGAC uvrD-EGFP-Sense 87 GGCCAGGGTATTAAATGGCTGGTGGCGGCATACGCCCGGCTGGAGTCGGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG uvrD-EGFP-AntiSense 72 AATGATTTTTTAGGCCAAATAAGGTGCGCAGCACCGCATCCGGCAACGTTATAGTGAACCTCTTCGAGGGAC uvrD Sense Primer 19 GAAGGCAGCGGTGAGCATA uvrD Antisense Primer 24 CATGACGTTGCAATTTATTGAATC valS-EGFP-Sense 87 GCGGAAGCGAAAGCGAAACTGATTGAACAGCAGGCTGTTATCGCCGCGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG valS-EGFP-AntiSense 72 AAAAGGCCGGAGCATGCTCCGGCCTTCGTTTTCATCACTGTGTTTTGATTATAGTGAACCTCTTCGAGGGAC valS Sense Primer 19 CATCGCGAAAGAGCGTGAG valS Antisense Primer 21 AGCCAGTATTTCACGGGGAGT visC-EGFP-Sense 87 CAACTTATCCGCCAGGCAATGGGATTAAACGATTTGCCTGAATGGCTGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG visC-EGFP-AntiSense 72 GGAGATGAAAGTGTGATGGGTATCAAATAAACAACAGAGGAGAAATTTTTATAGTGAACCTCTTCGAGGGAC visC Sense Primer 19 TTTGAAACTGGCCGACACG visC Antisense Primer 20 AATGAGGAAAATCTCCCGGC xerC-EGFP-Sense 87 CACCTTGCCTCGGTGTACGATGCGGCGCATCCACGCGCCAAACGGGGGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 228 xerC-EGFP-AntiSense 72 GGTCAAAGGTGAGCGCCGAGATGCGCCCCAAAGGCCGGTAAAAACGCATTATAGTGAACCTCTTCGAGGGAC xerC Sense Primer 24 CTCCACCACGCAAATCTATACTCA xerC Antisense Primer 22 CGGACGGTTATCGTAAAGGGTA xerD-EGFP-Sense 87 GTCGCTACCGAGCGTCTGCGGCAACTTCATCAACAGCATCACCCGCGGGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG xerD-EGFP-AntiSense 72 AAACAACATAAAACCTTTCTTCATAAATCTTCCCGTTCTTTTCAGACATCATAGTGAACCTCTTCGAGGGAC xerD Sense Primer 20 ACAGCGATCTCTCCACCACG xerD Antisense Primer 22 TGGCTAACGTTTGTTGAATTGC xseA-EGFP-Sense 87 AGTGAAGTAAAAAACATCCAGCCAGTAAAAAAATCGCGTAAAAAGGTGCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG xseA-EGFP-AntiSense 72 GCACGGGCATGGCTTGATATCGAAAAAACGCGTTGAATTCGTGCTGGCTTATAGTGAACCTCTTCGAGGGAC xseA Sense Primer 23 AATGCTAACCACACGTCTGGAAG xseA Antisense Primer 25 AGCCTGTGGTGCAGTAGATTACTTC xseB-EGFP-Sense 87 CTGTCTGACAATGAAGACGCCTCTCTAACCCCTTTTACACCGGACAATGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG xseB-EGFP-AntiSense 72 CCTGGTTGGCCTGCTTAACGCAGGCTTCGAGTTGCTGCGGAAAGTCCATTATAGTGAACCTCTTCGAGGGAC xseB Sense Primer 21 GCCAAATTACAACAAGCCGAA xseB Antisense Primer 21 GCCATACTGCATGGTTTCGAC xthA-EGFP-Sense 87 AGCATGGAAAAACCGTCCGATCACGCCCCCGTCTGGGCGACCTTCCGCCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG xthA-EGFP-AntiSense 72 CTCAAGGTTAATTCTCCTGACCCAGTTTGAGCCAGGAGAGCTGCTAAATTATAGTGAACCTCTTCGAGGGAC xthA Sense Primer 22 AAACCGGCATCGACTATGAAAT xthA Antisense Primer 23 ACGCAGAGACGGTATAACAAAGG yabC-EGFP-Sense 87 AACCCTCGTGCCCGTAGTTCAGTTCTGCGTATTGCAGAGAGGACGAATGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yabC-EGFP-AntiSense 72 TTCCCATCGATCCTTTAACTTTGCTTAGAGCTTCTGTCACTCTGCTGATCATAGTGAACCTCTTCGAGGGAC yabC Sense Primer 23 CTGCGAGCACTAGGCAAGTTAAT yabC Antisense Primer 21 CGATAACACCAGGCAATGCAT yacE-EGFP-Sense 87 CACGCACACTATTTGCAGCTTGCGTCGCAGTTTGTCTCACAGGAAAAACCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yacE-EGFP-AntiSense 72 TACGCATTTTTTCATTTAGTGGATGTTCAAAAAGGACCTGGGTCTGCATTATAGTGAACCTCTTCGAGGGAC yacE Sense Primer 20 TGCTATCGCATCGGATGTTG yacE Antisense Primer 23 ACGATGGGTAAATTAACGGTGAG yadF-EGFP-Sense 87 CGTTACCGTCACGGGATTTCCAACCTCAAGCTGAAACACGCCAACCACAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 229 yadF-EGFP-AntiSense 72 ACGAGTAAGTGTGAAGTTGCCGGATGTGTTGCATCCGGCATGGCATTTTTATAGTGAACCTCTTCGAGGGAC yadF Sense Primer 20 CTTGCTGCGTGATCTGGATG yadF Antisense Primer 22 GCCGTATATCGGCAAAGTGATT yadG-EGFP-Sense 87 CTGGAAGAGCTGTTTGTTTCACTGGTTAATGAAAAACAAGGAGATCGCGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yadG-EGFP-AntiSense 72 GGATCTCTTTCGCCCAGATGCTTTTTAGCGCCACCCAGTAAAGATGCATCATAGTGAACCTCTTCGAGGGAC yadG Sense Primer 27 CAGGTATTAAGTATGCGTAACAAAGCT yadG Antisense Primer 22 CTGCACCCAGATACGCATAAAG yadH-EGFP-Sense 87 TTTTATTTGATCTGTTGGTCGCTGATCCAACGTGGACGTGGTTTGCGTAGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yadH-EGFP-AntiSense 72 TAGCCGTCAAAACTCCTCTCCCCCAAATCCAGAGGAGAGGAAATAGCCTTATAGTGAACCTCTTCGAGGGAC yadH Sense Primer 20 CCTTTGGCGTACTGGTGGTC yadH Antisense Primer 18 CGCCTTCCTCCTTAGCCG yadR-EGFP-Sense 87 ACCAACCCGAACGCGAAAAGCACCTGCGGTTGCGGTTCTTCCTTTAGTATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yadR-EGFP-AntiSense 72 GAAGAGAGAAGATGTGCCGGATAGTTTATCCGGCACATGAACAACAGATTATAGTGAACCTCTTCGAGGGAC yadR Sense Primer 24 TTATACCGAAGGTCTGGAAGGTTC yadR Antisense Primer 19 CGTTTGCGCTGGATGAGAA yaeC-EGFP-Sense 87 GTTTACGAAGCAGCAAACAAAGTGTTTAACGGCGGAGCTGTTAAAGGCTGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yaeC-EGFP-AntiSense 72 AAGCGCCCGTCCTGAATGATATTACAAATTGTGGAAACAGCCTAAAAATTAATAGTGAACCTCTTCGAGGGAC yaeC Sense Primer 23 TCGTCCAGGCTTATCAGTCTGAC yaeC Antisense Primer 24 GGAGCTTGTAAAAATGACAAGACG yaeN EGFP-Sense 87 GTGGCTGAAGGTGAGAATGGCGTAAGTTTTGTCTGGCAGAAAACGCTTAGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yaeN EGFP-Antisense 72 TTGACTTTCGCCGGATGCGACGTTTGATGCGTCTTATCCGGCTTTCACTTATAGTGAACCTCTTCGAGGGAC yaeN Sense Primer 21 GGGGTATTTGTGACGCAAGAA yaeN Antisense Primer 20 GGTACGGTGGTGGTAAGCGA yaeS-EGFP-Sense 87 GCTAATCGAGAGCGTCGTTTCGGCGGCACCGAGCCCGGTGATGAAACAGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yaeS-EGFP-AntiSense 72 TAACACAAAAGCAGATATCAGGCGATACTTCAGCAAAAGCGACCCCCATCATAGTGAACCTCTTCGAGGGAC yaeS Sense Primer 24 TTTCGATGAACAAGACTTTGAAGG yaeS Antisense Primer 19 CCAGCGTTACAATGGCGAA yaeT-EGFP-Sense 87 TACGATGGAGACAAGGCAGAACAGTTCCAGTTTAACATCGGTAAAACCTGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 230 yaeT-EGFP-AntiSense 72 GCCTAAAGTCATCGCTACACTACCACTACATTCCTTTGTGGAGAACACTTATAGTGAACCTCTTCGAGGGAC yaeT Sense Primer 20 TGGTGTTCTCCTACGCCCAG yaeT Antisense Primer 19 TGGCCCGGCGATCTTATAT yaiD-EGFP-Sense 87 GCAGCGTTAATTCAAAACCTGATTGAAGGATTAGGTGGCGAAGCACAACGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yaiD-EGFP-AntiSense 72 GATAAGACGCGGCAGGCGTCGCATCCGGCATTAAAGGAAAATCAGCAATTATAGTGAACCTCTTCGAGGGAC yaiD Sense Primer 24 ACGAAGATATCGACCGTGAAGATT yaiD Antisense Primer 18 TGCCTGGTGTGGCTTCGT yajC-EGFP-Sense 87 AAACGTGACTTCGTAGCTGCCGTCCTGCCGAAAGGCACCATGAAGGCGCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yajC-EGFP-AntiSense 72 CAAAGGATAACGGTTTAACACGGCAATTCCCTTAGGGAAAAATTTTAATTATAGTGAACCTCTTCGAGGGAC yajC Sense Primer 23 CGCTGAATGACACCACTGAAGTA yajC Antisense Primer 21 CACCACGATCAGCATGACGTA yajQ-EGFP-Sense 87 GTACGTGGTGGCGATCTCGGTCAGCCGTTCCAGTTCAAAAACTTCCGCGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yajQ-EGFP-AntiSense 72 CAGGCGTTCATGCCGCATCTGACATGAACAAAACGCACATAGTCGCGATTATAGTGAACCTCTTCGAGGGAC yajQ Sense Primer 21 TAACGGGCAAATCTCGTGATG yajQ Antisense Primer 25 TCTGAATTACAAACCTTTGTAGGCC ybaB-EGFP-Sense 87 GCCTCTGTATCCTCCGGAATGCAGCTGCCGCCTGGCTTTAAGATGCCGTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybaB-EGFP-AntiSense 72 GACAGCGCAGTGCTTCCATAAGCTGTGTTAACAGCGGGCTGGTTTGCATCATAGTGAACCTCTTCGAGGGAC ybaB Sense Primer 22 CGTCGTATTGAAGAAACGCAGA ybaB Antisense Primer 19 AAGCAGCGTGAACGCCATA ybaD-EGFP-Sense 87 AGTTTCGAAGATATCAAAGAATTTGGCGAAGAGATCGCGCGCCTGGAGGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybaD-EGFP-AntiSense 72 TTGCGCCAGCTTTAGCGCCCGCGCCATGTAATACTCGTCCTGCACGGCTTATAGTGAACCTCTTCGAGGGAC ybaD Sense Primer 24 CCTATATCCGTTTTGCCTCTGTCT ybaD Antisense Primer 19 TGGGATGCGTGGTAAAACG ybaV-EGFP-Sense 87 CCGGGGATGGGCAATTCGCTGGTGGAACGTAATCTGGCGGTATTAACCCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybaV-EGFP-AntiSense 72 TGGTATGACCTCTTCAGTCTGGCAAAAAATTGCCACTATGCAAATTAATTATAGTGAACCTCTTCGAGGGAC ybaV Sense Primer 25 GAAGAGTACGGTCCGTTTAAAACTG ybaV Antisense Primer 22 TGTTTGCATAGCGCAATAACCT ybaX-EGFP-Sense 87 GCCGATAAACCGACGGTGATGGCAGCGATGAAGCAGAAAACCGGGTTGAGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 231 ybaX-EGFP-AntiSense 72 TTTTGTGGTGCCGGATGCTCAAGCCGCATCCGGCGACACCCGGAATAATTATAGTGAACCTCTTCGAGGGAC ybaX Sense Primer 20 TAATTTACGCGCCAACGGTT ybaX Antisense Primer 23 GAAAAGCTGGAGCAGATGGTTAA ybbF-EGFP-Sense 87 TCAATGGTGAAAGTCACGGCGGATGACGTTGAGCTGATTCATTTTCCGTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybbF-EGFP-AntiSense 72 CAAGGAAAACGGTTGCGTGGCTGTGAAATCAGCAAAGTTGCGGGTTTTTTATAGTGAACCTCTTCGAGGGAC ybbF Sense Primer 20 CCTGCTTTTCGCGTGGTACT ybbF Antisense Primer 20 GGGCACAGAGAATAGCACGG ybeA-EGFP-Sense 87 AGTCTGTACCGGGCGTGGAGCATCACCACCAACCATCCTTATCACCGTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybeA-EGFP-AntiSense 72 AGAGTTCTGTAGTTTCATCCGCTGCGTTTTCTACTCAAAGCTCCCTTATCATAGTGAACCTCTTCGAGGGAC ybeA Sense Primer 20 AGTTAGCCGCTGAGCTGGAA ybeA Antisense Primer 20 CGCGGACTCTGCCGTATAGT ybeB-EGFP-Sense 87 GTCATGCAGGAAGAGAGCCGTCGCCTGTATGAACTGGAAAAACTCTGGAGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybeB-EGFP-AntiSense 72 CCAGTCCGGCATTTTCGTTCCCACGGCGACAAGTTGCAGCTTCACGCATTATAGTGAACCTCTTCGAGGGAC ybeB Sense Primer 21 GATTTGGGCGATGTGATTGTC ybeB Antisense Primer 22 GACGCAGGTACTCGGTAAAACC ybeD-EGFP-Sense 87 GAAACACTGTATGAAGAACTGGGCAAAATCGATATTGTCCGCATGGTTCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybeD-EGFP-AntiSense 72 GAGGAGAGGGGAGTTACCCGACCAGGAGCCGGGTAACGGAGAAGCGAGTTATAGTGAACCTCTTCGAGGGAC ybeD Sense Primer 24 ACTATCAACGCCACTCATATCGAG ybeD Antisense Primer 24 AGTGGGGGAAAAGTATATCACAGC ybeX-EGFP-Sense 87 CAGGTTCATGTCAAAATCCCGGATGACTCACCCCAGCCGAAGCTGGATGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybeX-EGFP-AntiSense 72 TTCAATTAATGAGGCAAAAGCCATGTAGTTATCTATCCAGTTTCGGTTTTATAGTGAACCTCTTCGAGGGAC ybeX Sense Primer 18 TTCAAAGTGGCGATGGCC ybeX Antisense Primer 20 GGCACCGAATAATAACGCCA ybeY-EGFP-Sense 87 GAGATTATGCTTGCTCTGGGCTATGAGGATCCGTACATTGCCGAGAAAGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybeY-EGFP-AntiSense 72 CGTTAATCACCAACGGCGGGGACGTCTGCCAGTCAAATGCCTGGCAAATTATAGTGAACCTCTTCGAGGGAC ybeY Sense Primer 23 AGATGACGAAGCAGAAGAAATGG ybeY Antisense Primer 24 CATGTTAGTTAATCAAAACGCCGT ybeZ-EGFP-Sense 87 CGAAAAGCGGCGCTGGCAGCAGAACGCAAGCGCGAAGAACAGGAACAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 232 ybeZ-EGFP-AntiSense 72 ACCCGGAATTATCTTCACATGCCAGTTGTAAATCGAGGATCACCTGACTCATAGTGAACCTCTTCGAGGGAC ybeZ Sense Primer 24 GTATCGTTAACGCCTATGAAGCCT ybeZ Antisense Primer 22 GTCTGAAACTGGCTCTCTTCCG ybgC-EGFP-Sense 87 AAAATGAAGCCTCGTGCGCTTCCCAAGTCTATTGTCGCGGAGTTTAAGCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ybgC-EGFP-AntiSense 72 TAACCAGAAGGCTAGCCTTCAGGAACAAATCAAGGATATTCATGTCAGTCATAGTGAACCTCTTCGAGGGAC ybgC Sense Primer 23 GAAGCAGAGGTTCTGGTTGTTTG ybgC Antisense Primer 21 AATAATGGCCCAAGATGCGAT ycaJ-EGFP-Sense 87 CTCGCCTGGCTGGCTGAACAGGATCAAAATAGCCCCATAAAACGCTACCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ycaJ-EGFP-AntiSense 72 AGCCTGCGACCACAGGGATACAGTAACAACATTACCGCAACGATAACATTATAGTGAACCTCTTCGAGGGAC ycaJ Sense Primer 23 GCACAAACACGCTATTATTTCCC ycaJ Antisense Primer 24 TATCGAATTAAATGGGAGATGTGG ycbY-EGFP-Sense 87 GATTTCGCCCGTAACCGCCAGATCCACAACTGCTGGCTGATTACCGCAGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ycbY-EGFP-AntiSense 72 ACGACAGCCATGCGCCATGCATACTGATTAATGACATTACTATTCCTTTCATAGTGAACCTCTTCGAGGGAC ycbY Sense Primer 20 TTCCGTATGGATCTCGACGG ycbY Antisense Primer 21 GTTCTGCGTTATCGAGAAGCG yceG-EGFP-Sense 87 AACAAGTCTGTGCAGGATTATCTGAAAGTGCTTAAGGAAAAAAATGCGCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yceG-EGFP-AntiSense 72 CGCGCGGTAGTTTTGCCTGCGCCTTCCAGCCCCTCAATGACGATATACTTATAGTGAACCTCTTCGAGGGAC yceG Sense Primer 21 CCGATGGTAAAGGTGGTCACA yceG Antisense Primer 22 GTTCCCGAGTGAAAACCATGTC ycfC-EGFP-Sense 87 CGCCTGACCACTCAGGCAAAACAAATTCTTGCTCATTTAACCCCGGAGTTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ycfC-EGFP-AntiSense 72 GCGTCCATCGACAGGGGAAACGGCGGTCAGTGAGGATAATTCCATAGATCATAGTGAACCTCTTCGAGGGAC ycfC Sense Primer 21 CGGACGTCTGCAACTGATGTT ycfC Antisense Primer 22 TCAGCAAACCATATTCGCTGAA ycfF-EGFP-Sense 87 CACTTGTTGGGTGGCCGTCCGCTGGGACCAATGCTGGCGCATAAAGGTCTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ycfF-EGFP-AntiSense 72 GCAGTAACAACACCAGAGACACCAGCCCAAAGCATCCTTTTCTCATCGTTATAGTGAACCTCTTCGAGGGAC ycfF Sense Primer 23 ATTGCTGAGCAAGAAGGTATTGC ycfF Antisense Primer 21 AATTTCCGGATGTGAACGACA ycfH-EGFP-Sense 87 AACTTCGCCCGTCTGTTTCACATCGACGCTTCCCGCCTTCAATCCATCCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 233 ycfH-EGFP-AntiSense 72 GAACTTTACTCGTTTTAGCCATTAATTACGAGCTTTAAAAAAACTCATTCATAGTGAACCTCTTCGAGGGAC ycfH Sense Primer 20 GAACTGGCGCAGGTAACCAC ycfH Antisense Primer 21 GGTTATTCACCGCCCAATTTT ycfO-EGFP-Sense 87 ACCCGTCTGAATCAGTTACATGAACGCTGGCAGGAAGAGAAAGCAGGTCACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ycfO-EGFP-AntiSense 72 TCAAAACCGTGTAAATAGATAATCATCGCTTCCTCACATAAGCCAGGGTTATAGTGAACCTCTTCGAGGGAC ycfO Sense Primer 19 TCGCGACAGGAACTGATGG ycfO Antisense Primer 20 TCTCGTGGTTACCCGGACTG ycfV-EGFP-Sense 87 GAAATGCGTGATGGTCGTCTGACGGCGGAACTGAGCCTGATGGGGGCGGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ycfV-EGFP-AntiSense 72 GTCCGCGACTAAAACGCAGGCCAATTAATAACGATAAAGGCATCGCCATTATAGTGAACCTCTTCGAGGGAC ycfVSense Primer 21 GCAACTGGCGAAACGTATGAG ycfV Antisense Primer 20 ACGGAGATCAGCGACACCAT ycfW-EGFP-Sense 87 CCGGCGCGGCGCGCCAGTAATATTGACCCTGCGCGAGTCCTTAGCGGCCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ycfW-EGFP-AntiSense 72 ATATCAAACCCGTAATACATTGCCGCTCCTTGTTTTAATGTACTGCCTTTATAGTGAACCTCTTCGAGGGAC ycfW Sense Primer 21 CTGGTCACAGCATTGTTGCTG ycfW Antisense Primer 18 AAACACGCCAAGCGCAAT ychB-EGFP-Sense 24 GGCTTTGTGGCGAAAGGCGCTAATCTTTCCCCATTGCACAGAGCCATGCTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ychB-EGFP-AntiSense 72 AACGTCTGGAACAAGGTGACGTTGTCACCGAAACTCAGCTTGCCCGGCTTATAGTGAACCTCTTCGAGGGAC ychB Sense Primer 26 CTGAATTTGATACAGAGTCTGAAGCC ychB Antisense Primer 21 CAGGCGGTGTATTAAAGAGCG ychF-EGFP-Sense 87 AAAGATTACATCGTGAAAGATGGCGATGTGATGAACTTCCTTTTCAACGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ychF-EGFP-AntiSense 72 TTGCGCTCTTCATGAGATTTAGCGAAACCTCATGAGACAATAAATTAATTATAGTGAACCTCTTCGAGGGAC ychF Sense Primer 25 TCATCACTTACAAAGGTGAACAAGG ychF Antisense Primer 25 GAATGATTAGGACTCGGTGAAATGA yciA-EGFP-Sense 87 AAGTATGTCGCGGTTGATCCTGAAGGAAAACCTCGCGCCTTACCTGTTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yciA-EGFP-AntiSense 72 ATTCAGTAAGCAGAAAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTATTATAGTGAACCTCTTCGAGGGAC yciA Sense Primer 22 GCAACGCTATAAAGCGACAGAA yciA Antisense Primer 20 AGGCAGTGGGATTGTGGTGA yciB-EGFP-Sense 87 TTGTTAAGCGGTATCTATATCTACCGCCACATGCCGCAGGAAGATAAATCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 234 yciB-EGFP-AntiSense 72 GGAGAACAGAGGAGATACAGCGCCAGCCCCGAAGGACTGGCAGTCTGGTTATAGTGAACCTCTTCGAGGGAC yciB Sense Primer 24 TCAACTTTAAAGTCTTTGGCCTGA yciB Antisense Primer 21 CAGGCGCGATGCTACTATGAT yciO-EGFP-Sense 87 GATACGCCGGTCGTGGTGCGTGAAGGCGTAGGTGATGTGAAGCCTTTCTTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yciO-EGFP-AntiSense 72 GCAGCGAAACTTCCCTGCCATAGTCTTCCATCCGTGGAACGCAATTTGTTATAGTGAACCTCTTCGAGGGAC yciO Sense Primer 23 GAAACCGACAACGGTTATTGATC yciO Antisense Primer 21 ACAGCCCTGCTATCAACAGCA ydaO-EGFP-Sense 87 GAAGATGAAAATCAGTTGGATGAGTTACGGCTGAATGTGGTTGAAGTGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ydaO-EGFP-AntiSense 72 CCTCCATAAGAGTAGCCCGATACGCTTGCGCATCGGGCGCTATCCTGGTTATAGTGAACCTCTTCGAGGGAC ydaO Sense Primer 21 CGCGAAGAGATCCCACTACAA ydaO Antisense Primer 20 CATCTTATTCGGGATTGCCG ydgM-EGFP-Sense 87 TGGGATCTGAACACCATTCCCGTGCGTATCATTCCCGTGGAACACCATGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ydgM-EGFP-AntiSense 72 CCGCCGTTGAAATCCCAGATTTTATTTTTTCTGAATGCAGAGAATAACTTATAGTGAACCTCTTCGAGGGAC ydgM Sense Primer 19 CGGTCGCAGAAACACCTGA ydgM Antisense Primer 20 CTGGGTTTTCATCTCCGGTG ydhD-EGFP-Sense 87 CTGATCAAAGAAACTGCCGCTAAATACAAGTCTGAAGAGCCGGACGCGGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ydhD-EGFP-AntiSense 72 AAAGGACTGACGGATGCCCGTTCGCATCCGTCAGTATTGCAGGACGGATTATAGTGAACCTCTTCGAGGGAC ydhD Sense Primer 22 TCGTGATCGAAATGTATCAGCG ydhD Antisense Primer 20 TTTCAAGTTCGCCACAAGGG ydhH-EGFP-Sense 87 ACTGGCGCAAGCCAGGAGACGGTACTGGGGGCTATTTTCCCCGCTAACCCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ydhH-EGFP-AntiSense 72 CCAAGTGTAACGGCAGACGCGGTAAGAAAAATTCAGTTAACTCTGATATCATAGTGAACCTCTTCGAGGGAC ydhH Sense Primer 22 GGGATTACCAGGAAATCTGCCT ydhH Antisense Primer 20 CTGGTCTGGAGGGCAATACG ydjA-EGFP-Sense 87 GCATCTACGTCGATTAACGTCCCGGACCCGACGCCGTTTGTAACTTATTTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ydjA-EGFP-AntiSense 72 TGAATTCCTGCGCCTTTGCTCACAATCCAGACAGTTTCGCGACAATTATCATAGTGAACCTCTTCGAGGGAC ydjA Sense Primer 20 TTCTCTACCTCGGTACGCCG ydjA Antisense Primer 24 CCTCATTCCGTGAAGTGATAAGTG yeaZ-EGFP-Sense 87 CCGGTTTATTTACGTAACAACGTCGCATGGAAGAAACTTCCGGGCAAAGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 235 yeaZ-EGFP-AntiSense 72 AACCGCCATGGTGCGACTCCTTTTTTTCTCAGGGCATACTCTTAAGATTCATAGTGAACCTCTTCGAGGGAC yeaZ Sense Primer 21 TTTGCTGAGGGTAAAACGGTG yeaZ Antisense Primer 23 CCTGCCAGTATGCCTTTGATAAC yebC-EGFP-Sense 87 CAGGAAGTTTACCATAACGGTGAAATCTCTGATGAGGTCGCAGCGACTCTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yebC-EGFP-AntiSense 72 AGAATAATAGCCATCACGCGTCTCCGTTTTGCTGTTTAGCAGGCCTCATCATAGTGAACCTCTTCGAGGGAC yebC Sense Primer 21 ATATGCTGGAAGATTGCGACG yebC Antisense Primer 22 GACAGTTGCCTACCTACCTGGC yfcB-EGFP-Sense 87 ACCAAAGAGCAGCTTATTGCCGCACGAGAACATTTCGCGATTTATAAAGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfcB-EGFP-AntiSense 72 GTGTTTCCAGCCATCACGGCTCCGTTATTGTTGTGTTTGCGTGTTTACTTATAGTGAACCTCTTCGAGGGAC yfcB Sense Primer 22 AATATCCGGATGTTCCGTTCAC yfcB Antisense Primer 22 GGTGGTTACGCGAAAGAGTTGT yfcH-EGFP-Sense 87 TTTGCGTTTCGCTGGTACGATTTAGAAGAGGCGCTGGCGGATGTCGTTCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfcH-EGFP-AntiSense 72 ATCCTGTAACACCGGGAGTTAACTGGCGGATGTTTGCTGTAAACCACATCATAGTGAACCTCTTCGAGGGAC yfcH Sense Primer 21 TGGTATTAGGTGGACAACGCG yfcH Antisense Primer 20 ACCAGACGATCTTATCGCGC yffB-EGFP-Sense 87 ATGCTGCTGGGTTTCAGTGATTCCAGTTATCAGCAATTTTTCCATGAGGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yffB-EGFP-AntiSense 72 GCGGCGAATAAGCTGTTGTGTCAGCTCAATAACCGGGCACGACATAGACTATAGTGAACCTCTTCGAGGGAC yffB Sense Primer 22 TATCAAACGTCCATTGCTCTGC yffB Antisense Primer 21 GCATCCTGCATCATCAGGACT yfgB-EGFP-Sense 87 CGTACCCTGCGTAAACGGATGCAGGGTGAAGCCATCGACATTAAAGCGGTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfgB-EGFP-AntiSense 72 ATAAAACATTACGCCACGGTACATAAAGTAACCGTGGCGTAATGGCTATCATAGTGAACCTCTTCGAGGGAC yfgB Sense Primer 22 GTGGTGATGATATCGATGCTGC yfgB Antisense Primer 20 AGTCAGCAAAAACGCACCGT yfgM-EGFP-Sense 87 ACTCCGGCACTGAGCGAAATGATGCAGATGAAAATTAATAATTTGTCCATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfgM-EGFP-AntiSense 72 GAAAGCAGTCCTGGCAGCAGTAATTTACGCAATTGCATCGGGTCCCTCTCATAGTGAACCTCTTCGAGGGAC yfgM Sense Primer 21 ATAAGCAAGGTGCGCGTAGTG yfgM Antisense Primer 20 AGCGAACAGCCGCTTAAAAG yfgO-EGFP-Sense 87 CTGATCAAAGCCGTGATTCATGCCTGGCCCGATGGGCAAATCGCGCAAGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 236 yfgO-EGFP-AntiSense 72 AGGAACCCTGAAAAATCAGCCCGGCGAAACAGTTCGTCGGGCTGAAGATTATAGTGAACCTCTTCGAGGGAC yfgO Sense Primer 22 TTTATCGGTGGTGATCTTCGGT yfgO Antisense Primer 23 CTGGTGAGTATGTGTTATCGGGC yfhC-EGFP-Sense 87 ATGCGCCGCCAGGAAATTAAAGCGCAGAAAAAAGCGCAATCCTCGACGGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfhC-EGFP-AntiSense 72 TTCTCCCTCTCTGCTGTTTTCCAGGAAAGGGAGTGAAGAGAAACAAAATTATAGTGAACCTCTTCGAGGGAC yfhC Sense Primer 22 CGTTGCTCAGTGACTTCTTTCG yfhC Antisense Primer 24 TCCCAGTCGTCATCAAACTATTTG yfhF-EGFP-Sense 87 ACCAACCCGAACGTCAAAGATGAGTGTGGTTGCGGCGAAAGCTTCCACGTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfhF-EGFP-AntiSense 72 AACAAACCCCACGCGCAGGCGACCACGGTGGGGTTATCGGTATGCGCATCATAGTGAACCTCTTCGAGGGAC yfhF Sense Primer 20 AAGAAGGCCTGAACGAAGGG yfhF Antisense Primer 27 GAGGGTGAAGTAATCCATAACATTCTC yfhL-EGFP-Sense 87 GAAGAACAGTTGTGGGATAAATTTGTGCTGATGCACCACGCGGATAAAATTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfhL-EGFP-AntiSense 72 TAAGGCTTCCCAGTAATATAATTAATACTCTACTTCCAGAGTAGAATATTATAGTGAACCTCTTCGAGGGAC yfhL Sense Primer 21 TGCCCGATCCCCAATACTATT yfhL Antisense Primer 20 CCAGTCGGACGCACCTTTAA yfhO-EGFP-Sense 87 GAAATGTACAAGCAGGGCGTGGATCTGAACAGCATCGAATGGGCTCATCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfhO-EGFP-AntiSense 72 AACTTTTTCGCTGTAAGCCATTATAAATTCTCCTGATTCCGATACCGATTATAGTGAACCTCTTCGAGGGAC yfhO Sense Primer 20 CGTCTGCGTGACCTTTCTCC yfhO Antisense Primer 21 TTACGCGGATTCTCGTAATGG yfhP-EGFP-Sense 87 GACGCGCCACGCACCCGCACACAAGACGCGATCGACGTTAAGTTACGCGCTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfhP-EGFP-AntiSense 72 GTGTTTACGGAGTATTTAGCACTCCGGCCTGATTCTGAATTCTTTTTATTATAGTGAACCTCTTCGAGGGAC yfhP Sense Primer 22 TAACCAGGAAGTGCTGGATGTG yfhP Antisense Primer 20 CAGGCTACCGGCTGGATGTA yfhQ-EGFP-Sense 87 CGCGGGATTCTGGCTTCTATTGAGCAGCAGAATAAAGGTAACAAGGCCGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfhQ-EGFP-AntiSense 72 ACTGTAGGCCTGATAAGACGCATTACGCGTCGCATCAGGCAACGGCTGTTATAGTGAACCTCTTCGAGGGAC yfhQ Sense Primer 23 GGAAAGCCAGGAGTTGAATATCC yfhQ Antisense Primer 21 GCCTTATCCGACCTACGGTTC yfiH-EGFP-Sense 87 TATCGTCGCGACAAGACCACCGGTCGTATGGCAAGTTTCATTTGGCTGATACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 237 yfiH-EGFP-AntiSense 72 TAATGTGAAAAGAAAATCACGCGTACCGGATCGTCTTGATTCTTTAGGTTATAGTGAACCTCTTCGAGGGAC yfiH Sense Primer 22 GCGACCGTTGTACATATACGGA yfiH Antisense Primer 24 AGGTCATCCCTCAATTATTCAAGG yfiO-EGFP-Sense 87 GCGCAAGCTGAAAAAGTAGCGAAAATCATCGCCGCAAACAGCAGCAATACACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfiO-EGFP-AntiSense 72 TAAAAAAACGGCAGCTCAAGGGCTGCCGTTTTGTGTTTCAGGTTTCTGTTATAGTGAACCTCTTCGAGGGAC yfiO Sense Primer 22 GCATACCGTCAGATGCAGATGA yfiO Antisense Primer 24 AATCGCTTCAGTTTCACAACTGAC yfjF-EGFP-Sense 87 GCCGATCCGAAAGAGCTTCGCAGGCAACGAGCAGAAAAATCAGCGAATAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfjF-EGFP-AntiSense 72 CTCAAAGACGTTAAAAAAGGTGCTCAATGAGCACCTTTTTTCTGTCTGTTATAGTGAACCTCTTCGAGGGAC yfjF Sense Primer 19 CGGGTGGAGATTTATCGCC yfjF Antisense Primer 23 AACCTGCGCTGAGTGGTAACTAA yfjG-EGFP-Sense 87 AATATGGTCCAGGCTTTTACGGTTCGTGCGAAAGAGGTTTACAGTGCCAGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yfjG-EGFP-AntiSense 72 TGCAGGTACTGCTTCTCAGGTAGCGCATAAGCCACCTCAACGGCAATTTTATAGTGAACCTCTTCGAGGGAC yfjG Sense Primer 20 ATTGAACTCGCCTTTGGTCG yfjG Antisense Primer 19 ATTCCAGCAAGCCACTGGC ygbB-EGFP-Sense 87 GGGGAAGGGATTGCCTGTGAAGCGGTGGCGCTACTCATTAAGGCAACAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygbB-EGFP-AntiSense 72 CGGTGCCTTGCGGTTTACCGTGGAGGTAAGTGAGATTATCAAACTCAATCATAGTGAACCTCTTCGAGGGAC ygbB Sense Primer 24 TTAACGTGAAAGCCACTACTACGG ygbB Antisense Primer 20 GTCTTCCGGATTGGCTTTCA ygbP-EGFP-Sense 87 TTGGCACTGGCCGAGTTTTACCTCACCCGAACCATCCATCAGGAGAATACACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygbP-EGFP-AntiSense 72 GGCCTTCACCGCCAAAGGCATGTACGTCAAAACCGTGTCCAATTCGCATTATAGTGAACCTCTTCGAGGGAC ygbP Sense Primer 19 AAGGCCGTGCGGATAACAT ygbP Antisense Primer 20 TGCGTACGCCACCAATGATA ygbQ-EGFP-Sense 87 CTGGTGCCTGACGCGTCGAAGCGCGCACAGTCTGCGGGGCAAAACAATCGACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygbQ-EGFP-AntiSense 72 GGCGCAAACATCCAAATGAGTGGTTGCCATGTTAATTCCCGGGCTGATTTATAGTGAACCTCTTCGAGGGAC ygbQ Sense Primer 23 CGTAATGAACTCAGCATGACCAG ygbQ Antisense Primer 21 TGCTTAGGACATTCCGTTTGC ygcA-EGFP-Sense 87 TTCCCACACACGGGACATCTGGAATCGATGGTACTTTTCTCGCGCGTTAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 238 ygcA-EGFP-AntiSense 72 TCGTCCTCTCCTTTAGGGACCAGACCTGCCGAAATCGGCAAATCGCAACTATAGTGAACCTCTTCGAGGGAC ygcA Sense Primer 22 AAAGCAGGATATACCATTGCGC ygcA Antisense Primer 24 ATTGATATGTGCACTTCTTACCGC ygdL-EGFP-Sense 87 GCGGTTTCTCATGCGCTGAAGAAGATGATGGCGAAAGCGGCGCGTCAGGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygdL-EGFP-AntiSense 72 ACAGAACCGGGTCGGATAAGACGTTCGCGTCTCATCCGACCTGATTGTTTATAGTGAACCTCTTCGAGGGAC ygdL Sense Primer 18 TGGTGACCGCCACCTTTG ygdL Antisense Primer 19 TATCGCTGCGACGAAGCAG ygdP-EGFP-Sense 87 GAAAATACGCCAAAACCACAAAACGCATCTGCTTATCGACGTAAAAGAGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygdP-EGFP-AntiSense 72 TACCTTTTCGACTATTTCGCGCAGGCGAGTGAGCATAATTGGCGTGACTTATAGTGAACCTCTTCGAGGGAC ygdP Sense Primer 21 AAAGAGTTCGCGAGTGTGGTG ygdP Antisense Primer 23 AATATTTAACGCCTCATTCAGGC ygfA-EGFP-Sense 87 TGGGATATCCCTCTTCCTGCGGTGGTTACACCGTCGAAAGTCTGGGAGTGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygfA-EGFP-AntiSense 72 GGTCAAAGAATCAACGATGTCAATCAGGGCGATGCGGGTGTATCGCCCTTATAGTGAACCTCTTCGAGGGAC ygfA Sense Primer 22 GCATGATTGTCAGTTGGTGGAA ygfA Antisense Primer 23 GCAATCCTCCCTTACCCTTACTC ygfB-EGFP-Sense 87 TTTACTCATCCGCAACCGACCGCGCCAGAAGTACAAAAACCGACTCTACACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygfB-EGFP-AntiSense 72 TCTTGCCGGGATATCTCACTCATAACACTCTCCTTACGTTTTTTGTTTTTATAGTGAACCTCTTCGAGGGAC ygfB Sense Primer 24 AAGAGATCATCGAATACGTTCGTG ygfB Antisense Primer 21 GGCGCTACGTGTTACTTCTGG ygfE-EGFP-Sense 87 GCGTTACTTGAACAAGGTCGCATCACCGAAAAAACTAACCAAAACTTTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygfE-EGFP-AntiSense 72 GAGAAATTTTGTCTTCACGGTTACTCTACCACAGTAAACCGAAAAGTGTCATAGTGAACCTCTTCGAGGGAC ygfE Sense Primer 19 TTCGGATGCTGCAGCAGAC ygfE Antisense Primer 22 CGCCACATTCTTGTGGTATGAA ygfY-EGFP-Sense 87 ATGGTCCGACTCATCCAGACACGGAACCGGGAACGTGGTCCTGTGGCAATCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ygfY-EGFP-AntiSense 72 CCATGAATCAGCAAGGAAAGCCACTGTGCGCGCCAGGAGACGCGCAAATCATAGTGAACCTCTTCGAGGGAC ygfY Sense Primer 24 CCTGTTTAACTGGCTGATGAATCA ygfY Antisense Primer 24 CATGAGTAAAATAACAGCGGCAAC yggH-EGFP-Sense 87 GGTCATCGTCTTGGTCACGGAGTATGGGACTTAATGTTCGAGAGGGTGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 239 yggH-EGFP-AntiSense 72 CGTCGATGTGCATTTTTTTACGCAGACGACGGCTACGGTTCTTTGCCATTATAGTGAACCTCTTCGAGGGAC yggH Sense Primer 25 TGTCAGAGAGCAATGATTACGTACC yggH Antisense Primer 21 GCCACCGAAAATCCTAATTCC yggJ-EGFP-Sense 87 ACTGCGCTCACCGCCATTACCGCGCTACAAGTACGATTTGGCGATTTGGGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yggJ-EGFP-AntiSense 72 TGCGATGGGGTCCATCACGATGCCGAGCTTGATCATTATTCTTCTCCGTTATAGTGAACCTCTTCGAGGGAC yggJ Sense Primer 21 GATATCCTGTTGGGACCTCGC yggJ Antisense Primer 19 GACGCTGTGCTTCCAGCAA yggR-EGFP-Sense 87 ATGATAACGTTTCAGCAGAGTTATCAGCACCGGGTGGGGGAAGGGCGTTTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yggR-EGFP-AntiSense 72 TTATTGCCTTTGTAGGCCCGATAAGCTTGCGCATCGGGCATGGCAACGTCATAGTGAACCTCTTCGAGGGAC yggR Sense Primer 20 CGAAGGGAAAACCCACCAGT yggR Antisense Primer 20 TCTGGCAGCGGCTATAGGTC yggS-EGFP-Sense 87 ATGGTTCGTATCGGCACTGCAATTTTTGGTGCGCGTGATTACTCTAAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yggS-EGFP-AntiSense 72 CCGTTGAAAGCAGGAAAGTCAACGTATTCATGGCGTTCCTTTAATTCCTTATAGTGAACCTCTTCGAGGGAC yggS Sense Primer 20 TGTCGGACGATATGGAAGCC yggS Antisense Primer 21 ATCACAATGAGCCCACTGCAT yggT-EGFP-Sense 87 GTATTACAGGCAACCGGAAATATGCTGCTGCCAGGGCTGTGGATGGCGTTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yggT-EGFP-AntiSense 72 GAATATAGAGCCGTAAAACCAGACCGTCATCATTAACTGTTACGGCATTCATAGTGAACCTCTTCGAGGGAC yggT Sense Primer 24 GTATGTCATCAATATGGGTGTCGC yggT Antisense Primer 22 AATAGAATCACGGCTGGCTTTC yggV-EGFP-Sense 87 TCCCACCGTGGTCAGGCGTTGAAACTGCTGCTGGACGCTTTACGTAATGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yggV-EGFP-AntiSense 72 TTCTGCACGCACCACGGGATGTGAATGTAGAGACTCAGCGGAGGTAATTTATAGTGAACCTCTTCGAGGGAC yggV Sense Primer 22 TACCTTCCGAAGGGAAAACCGC yggV Antisense Primer 21 GCGTGAGAGTTGAAATCGCAG yggW-EGFP-Sense 87 GAACATGGGAAGCTGTTTTTAAATTCGCTGCTGGAGCTTTTTCTGGCTGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yggW-EGFP-AntiSense 72 CACATGTCGGCTGGATAAGGCGTTCACGCCGCATCCGGCAATACAAGTTTATAGTGAACCTCTTCGAGGGAC yggW Sense Primer 21 TATCTCACCGAATGTGCGGAT yggW Antisense Primer 23 GCGCGTCTTATCAGACCTACAAC yggX-EGFP-Sense 87 TTCGAGGGTAAAGAGGTGCATATCGAGGGCTATACGCCGGAAGATAAAAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 240 yggX-EGFP-AntiSense 72 TGTGTTTGTTTTTATGCAAGTTGCCGGAGGCGTGCTCCGGCACTGTTTTTATAGTGAACCTCTTCGAGGGAC yggX Sense Primer 20 GCTGCTTGAGCAGGAGATGG yggX Antisense Primer 24 AGATATTTTTTCATCATTCCGGGT yhbC-EGFP-Sense 87 GAAGTGTTCGCGCTGAGTAATATCCAGAAGGCGAACCTGGTTCCCCACTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhbC-EGFP-AntiSense 72 AGCCAAAATTTCTTTGTTCATCGCGGGCTTTTCACCTCATCCAGACTATTATAGTGAACCTCTTCGAGGGAC yhbC Sense Primer 24 GATCACAGTTACCGTCGAAGGTAA yhbC Antisense Primer 22 TTTTCATTGGATACGGCTTCAA yhbG-EGFP-Sense 87 TTACAAGACGAACACGTTAAGCGTGTATACCTTGGGGAAGACTTCAGACTCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhbG-EGFP-AntiSense 72 TGTTCAGAATCGTACTCTCCTGCTAAAACGTCGCAAACTTCTACCCTATCATAGTGAACCTCTTCGAGGGAC yhbG Sense Primer 22 AACGCGCTTATATCGTCAGTCA yhbG Antisense Primer 23 CTAAGCCTGAGTTGCAAACCTTG yhbJ-EGFP-Sense 87 CGCGGTAAAAACGTCCAGTCACGCCATCGTACGCTGGAAAAACGTAAACCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhbJ-EGFP-AntiSense 72 GGGCATGCATGCCCAGCTTGTTTGTGATTTCAACAGTTTGCTTGACGGTCATAGTGAACCTCTTCGAGGGAC yhbJ Sense Primer 22 CACCGTTCGGTGTATATTGCAG yhbJ Antisense Primer 23 AATTCAAACAGCTTCATTGCAGG yhbN-EGFP-Sense 87 CTGCAGGACAAAAACAACAAAGGCCAGACCCCGGCACAGAAGAAGGGTAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhbN-EGFP-AntiSense 72 TTTATAGGCTTTTGCAAGGTTCTTTGCAGTTAATGTTGCCATAACGAATTATAGTGAACCTCTTCGAGGGAC yhbN Sense Primer 20 AAAATGCAGGCTTTCAGCGA yhbN Antisense Primer 20 GTTGACGGTCAGGCTGACGT yhbY-EGFP-Sense 87 ACGCTGGTGCTTTATCGCCCAACTAAAGAACGTAAAATCTCGCTGCCACGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhbY-EGFP-AntiSense 72 GAAAACCCCTCGTTTTACACAGCAAATGTGTGTAACTTTAGGATAATCTTATAGTGAACCTCTTCGAGGGAC yhbY Sense Primer 21 TGATCGTGGAAGCTATCGTGC yhbY Antisense Primer 20 GAGCGTGGCATTTTGCTCTC yhbZ-EGFP-Sense 87 GACGACTGGGACGAAGACGACGAAGAAGGCGTTGAGTTCATTTACAAGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhbZ-EGFP-AntiSense 72 CCTGATAAGCGTAGCGCATCAGGCTGATTTGGCGTTTATCATCAGTGATTATAGTGAACCTCTTCGAGGGAC yhbZ Sense Primer 24 TGAAGAGATTGCTGAAGAGGATGA yhbZ Antisense Primer 22 CTTATCCGGCCTACAAAATCGT yhdG-EGFP-Sense 87 GATGCCAGCGAACAGCTGGAGGCGTTGGAGGCATACTTCGAAAATTTTGCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 241 yhdG-EGFP-AntiSense 72 GAATTTACGCGTTGTTCGAACATAGTTCTGTCAGCTCTTTATTTCTGTTTATAGTGAACCTCTTCGAGGGAC yhdG Sense Primer 20 GGAACACGCTCCAAATGACC yhdG Antisense Primer 27 AGAGTTAACGGTAGAAACGGTCAGTAC yheS-EGFP-Sense 87 CTGGAAGCCCAGGAGCAGCTTGAGCAGATGCTGCTGGAAGGCCAAAGCAACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yheS-EGFP-AntiSense 72 CAGCACTGCTGCTGAATTCATTGGCATCGGTCGTCGTTATCTGCGCCATCATAGTGAACCTCTTCGAGGGAC yheS Sense Primer 24 GAACTGTATGACCAGAGCCGTAAA yheS Antisense Primer 20 GCAGCATGGTTTGCAGATGA yhgF-EGFP-Sense 87 GGTAATAGCGCGATGATGGATGCGCTGGCGGCGGCAATGGGCAAAAAACGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhgF-EGFP-AntiSense 72 CTGCCCTACGATTTCGTGCAAATTCGAACCGTAGGCCAGTACGGGCGTTTATAGTGAACCTCTTCGAGGGAC yhgF Sense Primer 25 AAGTGGATCTTCAGCGTAAACGTAT yhgF Antisense Primer 25 ATATTCTCCGGCACTTTTATGTCAG yhgH-EGFP-Sense 87 TTACGCAATGGTGCGGCGGCTGTCCAGGTCTGGTGCCTTTGTCGAACCTTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhgH-EGFP-AntiSense 72 TAATAGTTGACTATTTTAGTTGGTTATAATACGCCCATCATCGAGGCTCTATAGTGAACCTCTTCGAGGGAC yhgH Sense Primer 19 GCAGAGATTGCGCAGTTGC yhgH Antisense Primer 24 GCATCGGAAATACGGATCATAGTA yhgI-EGFP-Sense 87 AAAGGTGTGCGCGATCTCACCGAACACCAGCGCGGCGAACACTCCTACTACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhgI-EGFP-AntiSense 72 AATGACGCTTTAGCGTCGCATCGGGCAATCTACAAAAGAGGGGATAACTTATAGTGAACCTCTTCGAGGGAC yhgI Sense Primer 22 AGAAGCAGCTGCTGAACGAATT yhgI Antisense Primer 20 CGTCTGGCACATGTTCAACG yhhF-EGFP-Sense 87 GTGGCTTATCGGCTGTATCAACGCGAAGCACAAGGAGAAAGTGATGCTGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhhF-EGFP-AntiSense 72 TTGAGGATTAAAAATCCCCAAACGCAGAGCATTAACAAACGACCAATATTATAGTGAACCTCTTCGAGGGAC yhhF Sense Primer 24 TTCCAGCAAACTGGTCATTACATC yhhF Antisense Primer 19 ATCAGCGCCACGTTAACGA yhiN-EGFP-Sense 87 TGGTCGAGTGCGTGGGCTTGTGCGCAGGATTTGATTGCAGCAAAGTCGTCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yhiN-EGFP-AntiSense 72 AGCTCATTTTTAATGCGACTCTAATAATTTTCATCTTTAGGAAATAGGTCATAGTGAACCTCTTCGAGGGAC yhiN Sense Primer 21 TGGGGGGCTATAACTTCCAGT yhiN Antisense Primer 22 TCAGTTCACGGGCTCATTAGAA yhiR-EGFP-Sense 87 CTGGTTCCGGCAGGCACCGGGCACGCCACCGTAAGCTGGATCGTGCCGGAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 242 yhiR-EGFP-AntiSense 72 TACCGCGATTGTAGCGCGAGACGTAATAGGTGCCAGCAATGGCTGCAATTATAGTGAACCTCTTCGAGGGAC yhiR Sense Primer 25 TGGAAACTGGAACAACAGATGAATA yhiR Antisense Primer 24 CGATGGCGATGTAATCATAGTGTT yibK-EGFP-Sense 87 GTGTATGAAGCCTGGCGGCAGTTGGGGTATCCGGGAGCGGTATTGAGAGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yibK-EGFP-AntiSense 72 CTGTGATCGTGCCGGATGCGATGTAATCATCTATCCGGCCTACAGTAACTATAGTGAACCTCTTCGAGGGAC yibK Sense Primer 21 ATGAATCTGTCCAATGCGGTG yibK Antisense Primer 24 TTGAGTATGGGGATGGGATCTAAT yibN-EGFP-Sense 87 GAAGGCGTCGCTGGCTGGGCTGGCGAAAACTTGCCTTTGGTGCGCGGCAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yibN-EGFP-AntiSense 72 GGGCCTAATTGCAGCTAACGGCCTGCATCATGAAAGACGACAGGTAAATTATAGTGAACCTCTTCGAGGGAC yibN Sense Primer 20 ACGCACTGACGAAAGCTGGT yibN Antisense Primer 23 TGGAGTGTCCCCAGAAGCTTATA yibP-EGFP-Sense 87 GAAATTCGCCGCCAGGGTCAGGCGGTCAATCCACAGCCGTGGTTGGGAAGACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yibP-EGFP-AntiSense 72 CAACAGAGCGGCAAATGCAAGAACGTTACGACGAAATGGAAACAAAACTTATAGTGAACCTCTTCGAGGGAC yibP Sense Primer 21 CAGGGTCGGCCTTCACTCTAT yibP Antisense Primer 19 TGGCAAGTTTGCCAGCAAG yicC-EGFP-Sense 87 GAGCTGAAAGTGTTGATTGAGCAGATGCGCGAGCAGATTCAGAACATCGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yicC-EGFP-AntiSense 72 GAGTGGCGGCATTTGTGAAGGCGGAAAAGTAAGAATTGGCGTTACGAGTTATAGTGAACCTCTTCGAGGGAC yicC Sense Primer 23 ATCAATGCCGAAGTGACAAACTC yicC Antisense Primer 19 TTGCGGGGAAGAAAAATGC yidC-EGFP-Sense 87 TACCGTGGTCTGGAAAAACGTGGCCTGCATAGCCGCGAGAAGAAAAAATCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yidC-EGFP-AntiSense 72 AAAAAGGCGGTCAACTGACCGCCCTTATTTTAGCGAAAACTCACCGAATCATAGTGAACCTCTTCGAGGGAC yidC Sense Primer 23 CCTGGTAACCATTATTCAGCAGC yidC Antisense Primer 23 CATGATGTTCCTGTTGCTTTGTG yihA-EGFP-Sense 87 GATACCTGGTTTAGCGAGATGCAGCCTGTAGAAGAAACGCAGGACGGCGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yihA-EGFP-AntiSense 72 TTACGGGCCGGATACGCCACATCCGGCACAAGCATTAAGGCAAGAAAATTATAGTGAACCTCTTCGAGGGAC yihA Sense Primer 21 TTCGTTGAAGAAACAAGGCGT yihA Antisense Primer 23 CAGGCCAACGGTAGAATTGTAAT yihZ-EGFP-Sense 87 ATGCAGGTATCGCTGGTCAATGATGGCCCCGTGACATTCTGGTTGCAGGTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 243 yihZ-EGFP-AntiSense 72 ACATAGCGATACTCTCTCTTGTTTCCCGTGACAACCCTGGAAGCTGGCTCATAGTGAACCTCTTCGAGGGAC yihZ Sense Primer 19 CAAACAGGACGCTTCGCTG yihZ Antisense Primer 23 CCAGCGAAACTGATAGTAACGCT yjbN-EGFP-Sense 87 GCAGACATTAATGTGCTGGAACACGCGCTCAAACTGGTGGCGGATAAGCGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjbN-EGFP-AntiSense 72 GCGACGGTGCGCAGGGCGTGGTGAATTTGACTACTTTTTGGTGAAAAGTTATAGTGAACCTCTTCGAGGGAC yjbN Sense Primer 21 TTGGGCTTGTTCCAGGGTATT yjbN Antisense Primer 24 CTCTTGCTGGCATTACAAGAATCA yjeA-EGFP-Sense 87 CTGGGCGCGGAGACACTGGCTGAAGTCATCGCCTTTAGCGTTGACCGGGCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjeA-EGFP-AntiSense 72 GGCGCTTATCACGCCATTCTTCGCTGTTAATTCAGTAATTTTTCAGAATTATAGTGAACCTCTTCGAGGGAC yjeA Sense Primer 22 GTTGATCGTCTGGTGATGTTGG yjeA Antisense Primer 25 AAATATCCAGTCTTAAATTGTGGCC yjeE-EGFP-Sense 87 GTGAGTGCGGTTTCCTCTGCGGGTGAATTGTTGCTGGCGCGTTTAGCCGGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjeE-EGFP-AntiSense 72 TACCAACCAATTTCTGATGCGATACATCATCCCGCCACCTTTCAAAGGTTATAGTGAACCTCTTCGAGGGAC yjeE Sense Primer 24 ACACATTGATTATCAGGCACAAGG yjeE Antisense Primer 23 AACCTGAATATCAGAGAGCGTCG yjeK-EGFP-Sense 87 ATTGGCGGCGAACCCAGCAAAACGCCGCTGGATCTCCAGCTACGCCAGCAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjeK-EGFP-AntiSense 72 GCGTAGCGAATCAGGCAATTTTAATGTTTAACTTCCCTGTTTAATCAGTTATAGTGAACCTCTTCGAGGGAC yjeK Sense Primer 22 TATGCGTGAGTTGCTGACACTG yjeK Antisense Primer 21 TTTCAATGCACGATGTAGGCC yjeQ-EGFP-Sense 87 GAAAGCATGGCGCAGGTAAAAACGCGTAAAAACTTTTCTGATACGGATGACCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjeQ-EGFP-AntiSense 72 CGGATCCTGAAAAAAAGGGGACGATTCTAACGACGGTTAGCTTAATTGTCATAGTGAACCTCTTCGAGGGAC yjeQ Sense Primer 23 CCGTTTCGAAAACTATCACCGTA yjeQ Antisense Primer 18 TGTCGTTCCTGATCCGGC yjfH-EGFP-Sense 87 GTTTCGGTTGCGACCGGAATTTGCTTATTTGAAGCGGTGCGCCAGCGCAGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjfH-EGFP-AntiSense 72 CAAAAAAAAACCATCCAAATCTGGATGGCTTTTCATAATTCTGAGAAATTATAGTGAACCTCTTCGAGGGAC yjfH Sense Primer 22 ATTGCGATGAGTTGATCAGCAT yjfH Antisense Primer 21 GCCAACGGATTCCATGTCATA yjgA-EGFP-Sense 87 GAAATTGCGCTGATGTTGCCGGAGCTTCTGGAGCGTTGGATTGAACGCGTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 244 yjgA-EGFP-AntiSense 72 CCACGCTCAGATAACGTAGGTAGCGTTCTACATCGGTGTGTAAATCGGTCATAGTGAACCTCTTCGAGGGAC yjgA Sense Primer 21 TATCAACAAGGGCAAGGAACG yjgA Antisense Primer 22 TAAGCAGGGTTATCGGGCTAAG yjgF-EGFP-Sense 87 CGTCTGCCGAAAGACGTGAAGATTGAGATCGAAGCGATCGCTGTTCGTCGCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjgF-EGFP-AntiSense 72 TTGTCATCAGACTTAATCCGGGCATGATAGCCCGGATTTCCATCAAGATTATAGTGAACCTCTTCGAGGGAC yjgF Sense Primer 21 CCTTCTTCACCGAACACAACG yjgf Antisense Primer 20 ATAAGCGTAGCGCATCAGGC yjgP-EGFP-Sense 87 ACCGTGCCGGTCCGCCGCCTGCGCGCCAGTTTTTCGCGTAAAGGAGCGGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjgP-EGFP-AntiSense 72 TGAAAATAGTTTTACCGATATAGCGGTCAAGTACGCCAAAAGGTTGCATCATAGTGAACCTCTTCGAGGGAC yjgP Sense Primer 24 TTTAGCGATTGTTCTCAACCTTTG yjgP Antisense Primer 24 AGCATGAACAGTGTCATCATGATG yjgQ-EGFP-Sense 87 CCAAGCGCCAGCTTCTTCTTAATCAGCCTGTGGCTGTTAATGAGAAAATCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjgQ-EGFP-AntiSense 72 GCTAACAATAATAAAGGGAGCTTTCGCTCCCTTTATTCGTTCATTCGGTTATAGTGAACCTCTTCGAGGGAC yjgQ Sense Primer 21 GCTGACGTTGGTTTATGGCAT yjgQ Antisense Primer 22 TTTCAGGAATGAACGAAGCACA yjjK-EGFP-Sense 87 GGCGCAGACGCGCTGGAGCCGAAGCGTATCAAGTACAAGCGTATTGCGAAGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjjK-EGFP-AntiSense 72 TTTTGTAGGCCGGATAAGGCGTTCGCGCCGCATCCGGCATTTTACGCATTATAGTGAACCTCTTCGAGGGAC yjjK Sense Primer 25 GAGTTCTTCGAAGGTAACTTTACCG yjjK Antisense Primer 21 GACGCTTACCGCGTCTTATCA yjjV-EGFP-Sense 87 GAGATTGCGCAAGCGTTGCTTAATAACACGTATACGTTGTTTAACGTGCCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yjjV-EGFP-AntiSense 72 AGGCATTGTGCGCCAACTGCCGGATGCGGCGTGAACGCCTTATCCGGCCTATAGTGAACCTCTTCGAGGGAC yjjV Sense Primer 20 GATATGCCGCTCAACGGTTT yjjV Antisense Primer 24 GTAGGCATGATAAGACGCGTTAAG yleA-EGFP-Sense 87 ATTGCCCGTACCCGCAAAGAAAACGACCTTGGCGTGGGTTATTATCAGCCGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yleA-EGFP-AntiSense 72 GGATAAAGAGAGAAAAAACAAGGCCCACCGGAACGGCAGGCCTGAGAATTATAGTGAACCTCTTCGAGGGAC yleA Sense Primer 20 GTGGCAGAAACACCGGAATC yleA Antisense Primer 19 GGCGCAAGCATTGCAATAA yqcD-EGFP-Sense 87 TGGCGCAGTAATAGCGATTTTGTCCCATCGACCACAAGACTGGTTCGGCAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 245 yqcD-EGFP-AntiSense 72 CTTTCACAACCTTCTGCGTGAATCCAGCACGCAAAATTGAGAAAAAATTTATAGTGAACCTCTTCGAGGGAC yqcD Sense Primer 21 TACGCACGTTATACCCGTCGT yqcD Antisense Primer 22 TGATTACAATAGCCCTGCCTGA yqgE-EGFP-Sense 87 AAACTGATTGGTGTGGATATTCTCACCATGCCTGGTGTGGCAGGACACGCCCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yqgE-EGFP-AntiSense 72 CGCCAATGCTTTTGGTGCCGAAGTCGAAGGCGAGTAAGGTTCCACTCATCATAGTGAACCTCTTCGAGGGAC yqgE Sense Primer 23 CCGGCAGATCTGAATATTCTGTT yqgE Antisense Primer 18 GTGCCGGTAATGCGTTGG yqgF-EGFP-Sense 87 GACTCTGCCTCTGCGGTTATTATTCTCGAAAGCTATTTCGAGCAGGGATATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yqgF-EGFP-AntiSense 72 ATAAGACGCGATGATATTATTTACACTCCGCCAGGCGTTTAAATCGCCTTATAGTGAACCTCTTCGAGGGAC yqgF Sense Primer 20 TCCGGTCTGTTTGAACAGGG yqgF Antisense Primer 20 ATAAGGTGTTCACGCCGCAT yraL-EGFP-Sense 87 CACGGCGTGAAGAAAAATGCGCTGTATAAGTATGCGCTGGAGCAGCAGGGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yraL-EGFP-AntiSense 72 CGGTAAAAGAAGACGTTGCGTTTTGATTAACGCAACGCTTCGGCACTGTTATAGTGAACCTCTTCGAGGGAC yraL Sense Primer 20 GAACTGCCGCTGAAAAAAGC yraL Antisense Primer 20 CACAGCCACCATTACCGTCA yraN-EGFP-Sense 87 TTCACCGGGAATGAGGTTGAGTGGATTAAGGATGCCTTTAATGACCACTCACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yraN-EGFP-AntiSense 72 GTGAAGCAAGCTTTAATTCTTTCTTGCACGCTAATCCTTAAACCTTAATTATAGTGAACCTCTTCGAGGGAC yraN Sense Primer 23 AGTTTTGATACTGTGGATTGCCG yraN Antisense Primer 21 CCGCAATTTGAGTTTGAATGC yraO-EGFP-Sense 87 AATTGCCTGTGCGATCTGATCGATAACACGCTTTTCCCTCACCAGGATGATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yraO-EGFP-AntiSense 72 CGCGGAAATAAGGACTGCGATTGGCGATAATGCCTTCATGTATTCTCCTTATAGTGAACCTCTTCGAGGGAC yraO Sense Primer 21 CGTAGTGCTCGCATTCAGGAA yraO Antisense Primer 21 CACAGCAGCGGTACCCACTAC yrbA-EGFP-Sense 87 AAAGCGTATACCCCTGCGGAGTGGGCGCGCGATCGCAAACTGAACGGCTTTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrbA-EGFP-AntiSense 72 TGTTCTCAGTTAACAATTCATATCCGCTACCGGCGAATCGCCCATAGCTCATAGTGAACCTCTTCGAGGGAC yrbA Sense Primer 20 CGCATTCATGCTGTGTCGAT yrbA Antisense Primer 20 AATTGTGACTTCGCCCTGGA yrbD-EGFP-Sense 87 GCTGCTGCGCCAGGTAATAATGAAACCACTGAACCTGTGGGTACAACGAAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 246 yrbD-EGFP-AntiSense 72 CAAAGCGACCATCATTAAACGTTTAAACATGCGTCGGTTCTCCTGAAATTATAGTGAACCTCTTCGAGGGAC yrbD Sense Primer 18 AATAGTGGCGATGCGCCA yrbD Antisense Primer 23 TCCATCAGCTTATACGGATTGGT yrbE-EGFP-Sense 87 CTGGCTGTTCTGGGGCTGGATTTTGTGCTGACCGCATTGATGTTTGGGAATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrbE-EGFP-AntiSense 72 AATAAAAAGATACCCACCCAAATTTCATTTTTTTTCGTTTGCATGAACTCATAGTGAACCTCTTCGAGGGAC yrbE Sense Primer 19 ATTAGCCGGGCAACCACTC yrbE Antisense Primer 24 GTTCAGTACGTATGGACGTCACGT yrbF -EGFP-Senser 87 CCGTTCCGCTATCCTGCCGGCGATTATCACGCTGATCTTTTACCAGGGAGTCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrbF -EGFP-AntiSenser 72 ATCCCTTTATGTCCGAGCGACGCCAGCGCATTTAACAGCATGAGTGGCTTATAGTGAACCTCTTCGAGGGAC yrbF Sense Primer 22 TCAGTTTCTGGACGGGATAGCT yrbF Antisense Primer 19 CCGACCAGCGCATTGAATA yrbH-EGFP-Sense 87 CATTTACTCGGTGTGTTACATATGCATGATTTACTGCGTGCAGGCGTAGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrbH-EGFP-AntiSense 72 TCGCAAGCGACGCACCTGCTTTGCTCATTGTTGTTTATCCTTGAATCTTTATAGTGAACCTCTTCGAGGGAC yrbH Sense Primer 21 TATCACCTCCGTGATGGTTGC yrbH Antisense Primer 23 CAGCAGACGAATGTTCTCTGCTT yrbI-EGFP-Sense 87 TTATTACTCCTGGCGCAGGGCAAACTGGATGAAGCCAAAGGGCAATCGATACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrbI-EGFP-AntiSense 72 CCAGAACCGCCAGTGATAGCACAATGATAACCCAACGTCTGGCTTTACTCATAGTGAACCTCTTCGAGGGAC yrbI Sense Primer 19 ATTACGTGACGCGCATTGC yrbI Antisense Primer 20 CGGCCATATTAATGCCGATC yrdC-EGFP-Sense 87 AATCCTTCAGAAATCCGCGATGCCCTGACGGGTGAACTGTTTCGACAGGGGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrdC-EGFP-AntiSense 72 TTGCTGTGGGCTATCGGATTACCAAAAACAGCATAGGTTTCCATTATGTTATAGTGAACCTCTTCGAGGGAC yrdC Sense Primer 21 GATTGCCACCTTGTCGAACAG yrdC Antisense Primer 20 TTGCTGATGAATGAATGGCG yrfE-EGFP-Sense 87 GTCAGTGCGCTGTTCCTCGTGCGCGAATGGTTGAAAGGGCAGGGGCGAGTGCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrfE-EGFP-AntiSense 72 AACCCCATAAGCCTGATAAACATTGTGCATCAGGCAAACTTCACGCATTTATAGTGAACCTCTTCGAGGGAC yrfE Sense Primer 21 GACCCTGACTTCAATGAAGCG yrfE Antisense Primer 26 AAAATCGTGAAAACTCAATAAATTGC yrfH-EGFP-Sense 87 GACAAAAAAGAGCGCCGCGACCTGTTACGATTTAAACACGGCGACAGTGAACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG 247 yrfH-EGFP-AntiSense 72 ATGTAATTGGTCATGTTGCGGCATAATCATCTCTCTTGCAGGTGACAGTTATAGTGAACCTCTTCGAGGGAC yrfH Sense Primer 25 GTAAACTTAATGCCTTAACCATGCC yrfH Antisense Primer 20 CCGAAACGGTTACCAGTTCG yrfI-EGFP-Sense 87 GATATTGCTGAAATCCGCAACAACGCGTCTCCGGCAGATCCGCAAGTTCATCTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG yrfI-EGFP-AntiSense 72 TGTATTAGTCGTAAAATCCGGCAGAGCCCTCTCTGCCGGACATACTCATTATAGTGAACCTCTTCGAGGGAC yrfI Sense Primer 24 TGCGGTAACCACTATCTGTTCAAT yrfI Antisense Primer 22 CTGAATGAGTGACAAAGCGCAT ytfM-EGFP-Sense 87 GATAAAGACGAACACGGGTTACAGTTTTACATCGGTCTGGGGCCAGAATTACTCGAAGGTTCCGGTATGGTGAGCAAGGGCGAGGAG ytfM-EGFP-AntiSense 72 ACAGTAAGATAACGATAACCACGCCGAGGCTGATTTTTTTCCATAAACTCATAGTGAACCTCTTCGAGGGAC ytfM Sense Primer 21 CAATCAAACTCGATTTTGCCG ytfM Antisense Primer 18 CACCAGAAACGCCACCGA EGFP 3'-Forward Primer 21 GGTCCTGCTGGAGTTCGTGAC EGFP 5'-Reverse Primer 21 GTCGTCCTTGAAGAAGATGGT 248