Rapid Allelic Exchange in Enterohemorrhagic Escherichia coli (EHEC) and Other E. coli Using λ Red Recombination

Pamela J. Savage1, John M. Leong1, Kenan C. Murphy1

1 University of Massachusetts MedicalSchool, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 5A.2
DOI:  10.1002/9780471729259.mc05a02s00
Online Posting Date:  January, 2006
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Abstract

This unit describes an allelic exchange system for enterohemorrhagic E. coli (EHEC), and similar pathogenic species of bacteria. The phage λ Red recombination system is expressed from a plasmid, inducing a hyper‐recombinogenic state where electroporated PCR‐generated substrates recombine with the bacterial chromosome at high efficiency. The technique can be used to substitute a drug marker for the gene of interest, or used to generate a clean in‐frame deletion of the target gene. Single gene knockouts in EHEC, or deletions of whole pathogenicity islands, can be constructed. A procedure for the preparation of hyper‐recombinogenic electrocompetent cells is also described. Besides E. coli K‐12 and EHEC, this method has also been used for the construction of gene knockouts in enteropathogenic E. coli (EPEC), enteroaggregative E. coli, and uropathogenic E. coli, as well as Shigella flexneri and Salmonella enterica.

Keywords: E. coli; allelic exchange; gene replacement; recombineering; EHEC; electrocompetence; pathogenicity island; lambda Red; PCR

     
 
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Table of Contents

  • Basic Protocol 1: Red‐Promoted Gene Replacement in EHEC
  • Alternate Protocol 1: Generation of Unmarked Chromosomal Deletions in EHEC Using sacB Counterselection
  • Support Protocol 1: Generation of Electrocompetent Hyper‐Recombinant EHEC Carrying the λ‐Red Functions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Red‐Promoted Gene Replacement in EHEC

  Materials
  • Linear DNA substrate containing drug marker sequence (see )
  • Polymerase: Taq polymerase (New England Biolabs)
  • dNTPs ( appendix 2A)
  • Appropriate PCR buffer (e.g., see appendix 2A)
  • TE buffer ( appendix 2A)
  • Electrocompetent EHEC transformed with Red‐producing plasmid ( protocol 3)
  • SOC medium ( appendix 4A)
  • LB plates ( appendix 4A) containing appropriate antibiotic (see , )
  • Electroporator and 0.1‐cm gap length electroporation cuvettes
  • Additional reagents and equipment for PCR (Kramer and Coen, ), purification of DNA from aqueous solution (Moore and Downhan, ), and Southern blotting (unit 14.1; optional)

Alternate Protocol 1: Generation of Unmarked Chromosomal Deletions in EHEC Using sacB Counterselection

  • 3.5‐kb NotI fragment from pKM154 (i.e., the cat‐sacB cassette; Murphy et al., )
  • Restriction enzyme(s) and appropriate buffer
  • LB plates supplemented with chloramphenicol (LB/Cam; for antibiotic concentrations, see , )
  • LB plates without NaCl, supplemented with 10% sucrose (LB/Suc), room temperature and 30°C
  • LB plates supplemented with ampicillin (LB/Amp; for antibiotic concentrations, see , )
  • Linear DNA fragments containing precise (unmarked) deletion of the region of interest (Murphy et al., ) suspended in TE buffer
  • Additional reagents and equipment for constructing plasmids (Murphy et al., ), purifying DNA fragments by gel electrophoresis (Moore et al., ), and preparing electrocompetent hyper‐recombinant cells ( protocol 3, steps to )

Support Protocol 1: Generation of Electrocompetent Hyper‐Recombinant EHEC Carrying the λ‐Red Functions

  Materials
  • LB plates ( appendix 4A) containing 100 µg/ml ampicillin (LB/AMP)
  • EHEC containing pKM201 or pKM208 (Murphy and Campellone, )
  • SOC medium ( appendix 4A) containing 100 µg/ml ampicillin, 30°C
  • 100 mM IPTG (see recipe)
  • 20% (w/v) glycerol, 4°C
  • 30° and 42°C shaking water baths
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Figures

Videos

Literature Cited

Literature Cited
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   Campellone, K., Robbin, D., and Leong, JM. 2004. EspFU is a translocated EHEC effector that interacts with Tir and N‐WASP and promotes Nck‐independent actin assembly. Dev. Cell 7:217‐228.
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   Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.1.1‐2.1.10. John Wiley & Sons, Hoboken, N.J.
   Moore, D., Dowhan, D., Chory, J., and Ribaudo, R.K. 2002. Isolation and purification of large DNA restriction fragments from agarose gels. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.6.1‐2.6.12. John Wiley & Sons, Hoboken, N.J.
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