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

Abstract

Lambda Red recombineering is a DNA cloning and engineering technique involving recombination between homologous regions. The homologous recombination is mediated by the lambda Red genes consisting of red alpha, red beta and gam. Three lambda Red recombineering systems are currently available; the first is the plasmid-based system, in which lambda Red genes were cloned into temperature-sensitive plasmids; the second is the prophage-based system, in which lambda Red genes containing prophage were integrated into the Escherichia coli genome; the third is the integrative form system, characterized by the integration of lambda 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 lambda 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.

Salmonella spvC Gene Inhibits Pyroptosis and Intestinal Inflammation to Aggravate Systemic Infection in Mice

“…The primers used for bacterial strains construction are listed in Supplementary Table 1. STM-spvC was constructed using λRed recombination system (Song et al, 2010); and the corresponding plasmids were gifts from Professor Daoguo Zhou (Purdue University, West Lafayette, USA). pBAD/gIII expression system was used to construct STM-c-spvC K136A and STM-c-spvC (Szeliova et al, 2016).…”

A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies

“…In the case of homologous recombination, a single crossover between a targeting gene and a homologous DNA fragment on a chromosome can be realized with the aid of the endogenous RecA and RecBCD complex 6 7 8 . When the three genes gam , bet , and exo originating from λ phage, were employed, the targeting gene bordered with short-length (35–50 bp) homologous arms was more effectively integrated into the chromosome than the RecA-based recombination 9 10 11 12 . However, the recombination frequency dropped sharply when the targeting DNA exceeded 2.5 kb in size 13 14 .…”

An enhanced vector-free allele exchange (VFAE) mutagenesis protocol for genome editing in a wide range of bacterial species

“…In E. coli , homologous recombination, site-specific recombination, and transposon-mediated gene transposition techniques are used for chromosomal integration (Martinez-Morales et al 1999; Court et al 2002; Song et al 2010; Song and Lee 2013; Gu et al 2015). Among these, the VFAE method is considered the simplest and most straightforward protocol for bacterial genome editing and is based on the homologous recombination (Gomaa et al 2017).…”

“…The success that has been achieved with the two bacterial species E. coli BL21 and B. subtilis 168 showed that the VFAE protocol can be renamed as the “vectorless integrative-vector technique”. Thus, as long as the integrative vector (suicide vector) technique is functional with any bacterial strain, the VFAE protocol could be widely and successfully applied (Katzen et al 1999; Schweizer 2008; Song et al 2010; Xie et al 2011; Heap et al 2012; Sabri et al 2013; Wang et al 2015). …”