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.