Proceedings of the National Academy of Sciences · 2000 · 15.2K citations · 35 references
Life SciencesGeneticsMolecular GeneticsGenetic EngineeringMicrobial GeneticsRecombinationPlasmid BiologyPlasmid EngineeringPlasmid GeneticsPolymerase Chain ReactionRecombinant Plasmid ConstructionRecombination EnzymesAntibiotic Resistance GenesBacterial Plasmid ConstructionBacterial Recombination MechanismsEscherichia ColiFlp-frt Recombination SystemsInducible Promoter SystemsReca RecombinaseSynthetic Plasmids
We developed a simple, highly efficient method to disrupt *E. coli* chromosomal genes by using PCR primers that provide homology to target loci and demonstrated its utility by generating PCR products with 36–50‑nt extensions and antibiotic‑resistance plasmids flanked by FRT sites. The method relies on λ Red recombinase expressed from an inducible, curable plasmid to integrate PCR products bearing homology arms and antibiotic resistance flanked by FRT sites, followed by FLP recombinase from a helper plasmid to excise the resistance cassette.
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.
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Studies on transformation of Escherichia coli with plasmids
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