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

KA Datsenko, BL Wanner - Proceedings of the National …, 2000 - National Acad Sciences
KA Datsenko, BL Wanner
Proceedings of the National Academy of Sciences, 2000National Acad Sciences
We have developed a simple and highly efficient method to disrupt chromosomal genes in
Escherichia coli in which PCR primers provide the homology to the targeted gene (s). In this
procedure, recombination requires the phage λ 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 …
We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage λ 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, lacZYA, 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 procedure can be done in wild-type cells.
National Acad Sciences