Protocol

Strain Engineering

Volume 765 of the series Methods in Molecular Biology pp 27-42

Date:

Targeted Chromosomal Gene Knockout Using PCR Fragments

  • Kenan C. MurphyAffiliated withDepartment of Microbial and Physiological systems, University of Massachusetts Medical School Email author 

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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.

Key words

Recombineering Lambda red Gene replacement Strain development Electroporation Phage lambda Beta Exo Gam PCR