PCR-mediated gene replacement in Escherichia coli

The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCD Delta::red substitution. This technique allows for gene replacement in E. coli without prior cloning of the gene of interest. In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates. In other experiments, electroporation of recBCD Delta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation. The placement of lambda led and gain at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyperrec as one containing the lambda I ed for recBCD substitution. The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E, coli. Three of these replacements were performed without prior cloning of the genes. (C) 2000 Elsevier Science B.V. All rights reserved.