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Lambda Red-Mediated Recombination in Shiga Toxin-Producing Escherichia coli.

Kenneth G Campellone1, Alyssa M Coulter2

  • 1Department of Molecular & Cell Biology, Institute for Systems Genomics, University of Connecticut, Storrs, CT, USA. kenneth.campellone@uconn.edu.

Methods in Molecular Biology (Clifton, N.J.)
|March 11, 2021
PubMed
Summary

The Lambda Red recombination system efficiently engineers bacterial chromosomes for studying virulence factors in E. coli. This method allows for gene knockouts, large deletions, and markerless exchanges in bacterial pathogens.

Keywords:
Allelic exchangeBacterial geneticsEPECEnteropathogenic E. coliEscherichia coliHomologous recombinationLambda RedRecombineeringSTECShiga toxin-producing E. coli

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Bacteriophage Genetics

Background:

  • The Lambda (λ) "Red" recombination system is a powerful tool for bacterial genome engineering.
  • It has significantly advanced the study of bacterial pathogenesis, particularly in Shiga toxin-producing (STEC) and enteropathogenic E. coli (EPEC).

Purpose of the Study:

  • To describe strategies and procedures for Lambda Red-mediated genome engineering in STEC.
  • To highlight the utility of the Lambda Red system for studying virulence factors in pathogenic E. coli.

Main Methods:

  • Utilizing transient plasmid-driven expression of the Lambda Red system.
  • Employing homologous recombination between PCR-derived linear DNA and target loci in STEC/EPEC chromosomes.

Main Results:

  • The Lambda Red system facilitates efficient engineering of bacterial chromosomes.
  • Specific techniques include individual gene knockouts, deletion of large pathogenicity islands, and markerless allelic exchanges.

Conclusions:

  • Lambda Red-mediated genome engineering is a versatile and efficient approach for studying bacterial pathogens.
  • This methodology is crucial for advancing research in bacterial pathogenesis and virulence factor analysis.