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.
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Murphy, K.C. (2011). Targeted Chromosomal Gene Knockout Using PCR Fragments. In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_2
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DOI: https://doi.org/10.1007/978-1-61779-197-0_2
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