PCR-mediated gene replacement in Escherichia coli

The hyper-recombinogenic properties of an E. coli strain in which the recBC D genes have been replaced by lambda red recombination functions were explo ited in the development of a general PCR-mediated gene replacement scheme f or Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCD Delta::re d substitution. This technique allows for gene replacement in E. coli witho ut prior cloning of the gene of interest. In addition, the counter-selectab le marker sacB has been used to construct unmarked precise gene deletions w ithout the need to form sacB-containing plasmid integrates. In other experi ments, electroporation of recBCD Delta::red strains with high concentration s of linear DNA fragments (derived from plasmid digests) gave linear transf ormation rates approaching 1% of the survivors of electroporation. The plac ement 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 lam bda 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 recom bination in E, coli. Three of these replacements were performed without pri or cloning of the genes. (C) 2000 Elsevier Science B.V. All rights reserved .