Rapid generation of knock-out mutants in bacterial cells. Knock-in of genes for bacterial strain development. "Tagging" bacteria with visible genetic markers for environmental                localization studies. Direct sequencing of bacterial chromosomal DNA. EZ-Tn5™ Transposome™ complexes are formed between an EZ-Tn5™ Transposon and EZ-Tn5™ Transposase, and provide a simple and reliable method for generating a library of random gene knockouts in vivo.* Just electroporate the EZ-Tn5 Transposome into any of a broad range of living    bacterial cells and select for a marker encoded by the EZ-Tn5 Transposon (Fig. 1). Because there is no need for cell conjugation, suicide vectors, or specific host factors, EZ-Tn5 Transposomes are ideal for creating mutants in species that have poorly described genetic systems or lack adequate molecular tools. Ready-to-use EZ-Tn5 Transposomes* are available containing a kanamycin selectable marker (<KAN-2>).  This marker is readily expressed in many Gram-negative bacteria. You can also create your own EZ-Tn5 Transposome using one if            the EZ-Tn5 pMOD™ Transposon Construction Vectors and EZ-Tn5 Transposase.

Figure 1. The EZ-Tn5™ Transposon insertion site            in bacterial DNA can be sequenced directly using genomic DNA isolated            using the MasterPure™ Complete DNA Purification Kit and primers            homologous to the ends of the transposon.

All EZ-Tn5 Transposons contain unique primer-binding sites at each end for bidirectional sequencing. Hence, a gene knockout can be sequenced directly using bacterial genomic DNA as template and the primers provided with each Transposome. Transposon insertions made using an EZ-Tn5 <R6Kγori/KAN-2>Tnp Transposome Kit can be rescued and the flanking DNA sequenced.

EZ-Tn5 Transposome-mediated insertions have been made in many different microorganisms, including Acinetobactor, Campylobacter, Escherichia, Mycobacterium, Proteus, Pseudomonas, Saccharomyces, Salmonella, Trypanosoma, Xylella, and others. The number of transposition clones obtained is highly dependent on the transformation efficiency of the host cell (Table 1).

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Protocols for: EZ-Tn5™ Transposome™ Kits

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References:

1. Allsopp, L. P., et al. (2010) UpaH Is a Newly Identified Autotransporter Protein That Contributes to Biofilm Formation and Bladder Colonization by Uropathogenic Escherichia coli CFT073, Infect. Immun. 78 , 1659-1669.
2. Arrach, N., et al. (2010) High-Throughput Screening for Salmonella Avirulent Mutants That Retain Targeting of Solid Tumors, Cancer Res. 70 , 2165-2170.
3. Berger, C., et al. (2010) Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria, Microbiology 156 , 1963-1974.
4. Duffin, P. M. & Seifert, H. S. (2010) DNA uptake sequence mediated enhancement of transformation in Neisseria gonorrhoeae is strain dependent, J. Bacteriol. , JB.00442-10.
5. Guo, Y., et al. (2010) Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri, Appl. Envir. Microbiol. 76 , 2234-2242.
6. Hartmann, I., et al. (2010) Genes involved in Cronobacter sakazakii biofilm formation, Appl. Envir. Microbiol. , AEM.00930-09.
7. Jackson, L. A., et al. (2010) Transcriptional and Functional Analysis of the Neisseria gonorrhoeae Fur Regulon, J. Bacteriol. 192 , 77-85.
8. Johler, S., et al. (2010) Genes Involved in Yellow Pigmentation of Cronobacter sakazakii ES5 and Influence of Pigmentation on Persistence and Growth under Environmental Stress, Appl. Envir. Microbiol. 76 , 1053-1061.
9. Kouzuma, A., et al. (2010) Disruption of the Putative Cell Surface Polysaccharide Biosynthesis Gene SO3177 in Shewanella oneidensis MR-1 Enhances Adhesion to Electrodes and Current Generation in Microbial Fuel Cells, Appl. Envir. Microbiol. 76 , 4151-4157.
10. LeCuyer, B. E., et al. (2010) Genetic Characterization of the Nucleotide Excision Repair System of Neisseria gonorrhoeae, J. Bacteriol. 192 , 665-673.
11. Luke, N. R., et al. (2010) Identification and Characterization of a Glycosyltransferase Involved in Acinetobacter baumannii Lipopolysaccharide Core Biosynthesis, Infect. Immun. 78 , 2017-2023.
12. Qiao, J., et al. (2010) Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage {Phi}2954 infection, PNAS 107 , 6000-6004.
13. Rudolph, C. J., et al. (2010) RecG Protein and Single-strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity in Escherichia coli, Genetics , genetics.110.120691.
14. Tozzoli, R., et al. (2010) Production of the Subtilase AB5 Cytotoxin by Shiga Toxin-Negative Escherichia coli, J. Clin. Microbiol. 48 , 178-183.
15. Vanzin, G., et al. (2010) Characterization of Genes Responsible for the CO-linked Hydrogen-Production Pathway in Rubrivivax gelatinosus, Appl. Envir. Microbiol. , AEM.02753-09.
16. Yamanaka, K., et al. (2010) Identification and analysis of an {epsilon}-poly-L-lysine-degrading enzyme reveal the mechanism of {epsilon}-poly-L-lysine production and accumulation, Appl. Envir. Microbiol. , AEM.00853-10.
17. Zhang, J., et al. (2010) Promoting and Avoiding Recombination: Contrasting Activities of the Escherichia coli RuvABC Holliday Junction Resolvase and RecG DNA Translocase, Genetics 185 , 23-37.
18. Duval, V., et al. (2009) Combined Inactivation of lon and ycgE Decreases Multidrug Susceptibility by Reducing the Amount of OmpF Porin in Escherichia coli, Antimicrob. Agents Chemother. 53 , 4944-4948.
19. Ewing, C. P., et al. (2009) Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176, J. Bacteriol. 191 , 7086-7093.
20. Fricke, W. F., et al. (2009) Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family, J. Bacteriol. 191 , 4750-4757.
21. Kouvelis, V. N., et al. (2009) Complete Genome Sequence of the Ethanol Producer Zymomonas mobilis NCIMB 11163, J. Bacteriol. 191 , 7140-7141.
22. Lin, J., et al. (2009) Systematic identification of genetic loci required for polymyxin resistance in Campylobacter jejuni using an efficient in vivo transposon mutagenesis system, Foodborne Pathog Dis 6 , 173-185.
23. Porter, J. L., et al. (2009) Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum, Microbiology 155 , 1923-1933.
24. Ray, J. L., et al. (2009) Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome, Genetics 182 , 1165-1181.
25. Krishnan, K. & Flower, A. M. (2008) Suppression of {Delta}bipA Phenotypes in Escherichia coli by Abolishment of Pseudouridylation at Specific Sites on the 23S rRNA, J. Bacteriol. 190 , 7675-7683.
26. Pittman, G. W., et al. (2008) Assessment of Gut Bacteria for a Paratransgenic Approach To Control Dermolepida albohirtum Larvae, Appl. Envir. Microbiol. 74 , 4036-4043.
27. Rouws, L. F., et al. (2008) Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant, Arch Microbiol 189 , 397-405.

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For Research Use Only. EZ-Tn5™

* products covered by issued and pending patents.

Note: New EZ-Tn5™ Transposomes are being added regularly. Please visit Epicentre's® web site at www.epibio.com or subscribe to the Epicentre® Forum newsletter for information on new product releases.

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Applications

• Rapid generation of knock-out mutants in bacterial cells.
• Knock-in of genes for bacterial strain development.
• "Tagging" bacteria with visible genetic markers for environmental localization studies.
• Direct sequencing of bacterial chromosomal DNA

Benefits

• Rapid mutagenesis procedure is simpler and easier to use than chemical mutagenesis.
• More efficient than using mini-transposons with suicide plasmids.
• Broad host range: over 60 species of Gram-negative and Gram-positive bacteria transposed so far

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• EZ:TN5 Frequently Asked Questions