Rapid allelic exchange in enterohemorrhagic Escherichia coli (EHEC) and other E. coli using lambda red recombination

This unit describes an allelic exchange system for enterohemorrhagic E. coli (EHEC), and similar pathogenic species of bacteria. The phage lambda 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.