One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

摘要

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherhoia con in which PCR primers pro- vide the homology to the targeted gene(s). In this procedure, recombination requires the phage A Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, IacZYA, ompR-envZ phnR, pstB, pstCA, pstS, pstSCAB-phoU recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR- generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E coli and other bacteria because the proce- dure can be done in wild-type cells.