pq006640 - One-step inactivation of chromosomal genes in...

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Unformatted text preview: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products 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 pro- vide the homology to the targeted gene(s). In this procedure, recombination requires the phage l 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 proce- dure can be done in wild-type cells. bacterial genomics u FLP recombinase u FRT sites u Red recombinase T he availability of complete bacterial genome sequences has provided a wealth of information on the molecular structure and organization of a myriad of genes and ORFs whose functions are poorly understood. A systematic mutational analysis of genes in their normal location can provide significant insight into their function. Although a number of general allele replacement methods (17) can be used to inactivate bacterial chromosomal genes, these all require creating the gene disruption on a suitable plasmid before recombining it onto the chromosome. In con- trast, genes can be directly disrupted in Saccharomyces cerevisiae by transformation with PCR fragments encoding a selectable marker and having only 35 nt of flanking DNA homologous to the chromosome (8). This PCR-mediated gene replacement method has greatly facilitated the generation of specific mutants in the functional analysis of the yeast genome; it relies on the high efficiency of mitotic recombination in yeast (9). Directed disruption of chromosomal genes can also be done in Candida albicans by using similar PCR fragments with 50- to 60-nt homology extensions (10)....
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pq006640 - One-step inactivation of chromosomal genes in...

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