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Published online on May 30, 2000, 10.1073/pnas.120163297
PNAS | June 6, 2000 | vol. 97 | no. 12 | 6640-6645


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

(bacterial genomics / FLP recombinase / FRT sites / Red recombinase)

Kirill A. Datsenko and Barry L. Wanner*

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

Communicated by Jonathan Beckwith, Harvard Medical School, Boston, MA, April 11, 2000 (received for review February 13, 2000)

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 lambda  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.

* To whom reprint requests should be addressed. E-mail: BLW{at}bilbo.bio.purdue.edu.


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