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Homologous Recombineering to Generate Chromosomal Deletions in Escherichia coli

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The Bacterial Nucleoid

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1624))

Abstract

Homologous recombination methods enable modifications to be made to the bacterial chromosome. Commonly, the λ phage RED proteins are employed as a site-specific recombinase system, to facilitate recombination of linear DNA fragments with targeted regions of the chromosome. Here we describe methods for the efficient delivery of linear DNA segments containing homology to the chromosome into the cell as substrates for the λRED proteins. Combined with antibiotic selection and counterselection, we demonstrate that using this method facilitates accurate, rapid editing of the chromosome.

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References

  1. Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388

    Article  CAS  PubMed  Google Scholar 

  2. Mosberg JA, Lajoie MJ, Church GM (2010) Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186:791–799

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Datsenko KA, Wanner BL (2000) One step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair and engineering of chromosomal DNA using using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98:6742–6746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Herring CD, Glasner JD, Blattner FR (2003) Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli. Gene 311:153–163

    Article  CAS  PubMed  Google Scholar 

  6. Lee DJ, Bingle LEH, Heurlier K, Pallen MJ, Penn CW, Busby SJW, Hobman JL (2009) Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiol 9:252

    Article  PubMed  PubMed Central  Google Scholar 

  7. Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Stringer AM, Singh N, Yermakova A, Petrone BL, Amarainghe JJ, Reyes-Diaz L, Mantis NJ, Wade JT (2012) Fruit, a scar-free system for targeted chromosomal mutagenesis, epitope tagging and promoter replacement in Escherichia coli and Salmonella enterica. PLoS One 7:e44841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36

    Article  PubMed  PubMed Central  Google Scholar 

  10. Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97:5978–5983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bingle LEH, Constantinidou C, Shaw RK, Islam MS, Patel M, Snyder LAS, Lee DJ, Penn CW, Busby SJW, Pallen MJ (2014) Microarray analysis of the Ler regulon in enteropathogenic and enterohaemorrhagic Escherichia coli strains. PLoS One 9:e80160

    Article  PubMed  PubMed Central  Google Scholar 

  12. Li G, Young KD (2012) Isolation and identification of new inner membrane-associated proteins that localize to the cell poles in Escherichia coli. Mol Microbiol 84:276–295

    Article  CAS  PubMed  Google Scholar 

  13. Bryant JA, Sellars LE, Busby SJ, Lee DJ (2014) Chromosome position effects on gene expression in Escherichia coli K-12. Nucleic Acids Res 42:11383–11392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Moor H, Teppo A, Lahesaare A, Kivisaar M, Teras R (2014) Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF. Microbiology 160:2681–2693

    Article  CAS  PubMed  Google Scholar 

  15. Rufian JS, Sanchez-Romero MA, Lopez-Marquez D, Macho AP, Mansfield JW, Arnold DL, Ruiz-Albert J, Casadesus J, Beuzon CR (2016) Pseudomonas syringae differentiates into phenotypically distinct subpopulations during colonization of a plant host. Environ Microbiol 18(10):3593–3605

    Article  CAS  PubMed  Google Scholar 

  16. Mosberg JA, Yep A, Meredith TC, Smith S, Wang PF, Holler TP, Mobley HLT, Woodard RW (2011) A unique arabinose 5-phosphate isomerase found within a genomic island associated with the uropathogenicity of Escherichia coli CFT073. J Bacteriol 193:2981–2988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Haines S, Arnaud-Barbe N, Poncet D, Reverchon S, Wawrzyniak J, Nasser W, Renauld-Mongenie G (2015) IscR regulates synthesis of colonization factor antigen 1 fimbriae in response to iron starvation in enterotoxigenic Escherichia coli. J Bacteriol 197:2896–2907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Alsharif G, Ahmad S, Islam MS, Shah R, Busby SJ, Krachler AM (2015) Host attachment and fluid shear are integrated into a mechanical signal regulating virulence in Escherichia coli O157:H7. PNAS 112:5502–5508

    Article  Google Scholar 

  19. Jorgensen SB, Bojer MS, Boll EJ, Martin Y, Helmersen K, Skogstad M, Struve C (2016) Heat-resistant, extended-spectrum β-lactamase-producing Kelbsiella pneumonia in endoscope-mediated outbreak. J Hosp Infect 93:57–62

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK

    Jack A. Bryant

  2. Department of Life Sciences, School of Health Sciences, Birmingham City University, Birmingham, B15 3TN, UK

    David J. Lee

Authors
  1. Jack A. Bryant
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  2. David J. Lee
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Corresponding author

Correspondence to David J. Lee .

Editor information

Editors and Affiliations

  1. UMR CNRS 7241, INSERM U1050, CIRB, Collège de France, Paris, France

    Olivier Espéli

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Bryant, J.A., Lee, D.J. (2017). Homologous Recombineering to Generate Chromosomal Deletions in Escherichia coli . In: Espéli, O. (eds) The Bacterial Nucleoid. Methods in Molecular Biology, vol 1624. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7098-8_1

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  • DOI: https://doi.org/10.1007/978-1-4939-7098-8_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7097-1

  • Online ISBN: 978-1-4939-7098-8

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