title: Targeted chromosomal gene knockout using PCR fragments.
abstract: The development of recombineering technology has converged to a point that virtually any type of genetic modification can be made in the Escherichia coli chromosome. The most straightforward -modification is a chromosomal gene knockout, which is done by electroporation of a PCR fragment that contains a selectable drug marker flanked by 50 bp of target DNA. The phage λ Red recombination system expressed in vivo from a plasmid promotes deletion of the gene of interest at high efficiency. The combination of this technology with site-specific recombination systems of Cre and Flp has enabled genetic engineers to construct a variety of marked and precise gene knockouts in a variety of microbial chromosomes. The basic protocols for designing PCR substrates for recombineering, generating -recombineering-proficient electrocompetent strains of E. coli, and for selection and verification of recombinant clones are described.
authors.affiliation: Department of Microbial and Physiological systems, University of Massachusetts Medical School, Worcester, MA, USA. kenan.murphy@umassmed.edu
authors.forename: Kenan C
authors.initials: KC
authors.lastname: Murphy
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D005798:Genes, Bacterial / Q000235:genetics*; D016133:Polymerase Chain Reaction