Construction and functional characterization of an integrative form λ Red recombineering Escherichia coli strain

Abstract

λ Red recombineering is a DNA cloning and engineering technique involving recombination between homologous regions. The homologous recombination is mediated by the λ Red genes consisting of red α, red β and gam. Three λ Red recombineering systems are currently available; the first is the plasmid-based system, in which λ Red genes were cloned into temperature-sensitive plasmids; the second is the prophage-based system, in which λ Red genes containing prophage were integrated into the Escherichia coli genome; the third is the integrative form system, characterized by the integration of λ Red genes (or their counterparts) into the E. coli genome. In this study, a novel integrative form recombineering host, E. coli LS-GR, was constructed through the integration of functional recombineering elements including λ Red genes, recA, araC and aacC1 into the E. coli DH10B genome. LS-GR shows high recombination efficiency for medium copy number vector and single copy number BAC vector modifications. The results indicate that LS-GR could be used as a general recombineering host strain. © 2010 Federation of European Microbiological Societies.