coli genome. In this study, a novel integrative form recombineering host, E. coli LS-GR, was constructed through the integration of functional recombineering MG-132 ic50 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. λ Red recombineering (recombination-mediated genetic engineering) is an in vivo DNA cloning and engineering technique used primarily in Escherichia coli (Murphy, 1998; Zhang et al., 1998; Yu et al., 2000; Court et al., 2002; Sharan et al., 2009). The recombinases catalyzing the recombination between homologous DNA fragments are encoded by the λ bacteriophage red operon, where the exo (redα) gene PD0332991 price encodes a 5′3′ exonuclease, creating a single-stranded protruding overhang of DNA; the bet (redβ) gene encodes a single-stranded DNA-binding protein that promotes the annealing of two cDNA molecules;
and the gam (redγ) gene encodes the Gam protein that protects the incoming (modifying) DNA from being degraded by host endonucleases, RecBCD and SbcCD (Murphy, 1991). The length of the homologous region used for homologous recombination can be as short as 35–50 bp (Poteete, 2001; Court et al., 2002), which can be easily introduced through PCR primer synthesis, thus considerably facilitating the experimental process. λ Red recombineering is also an efficient
gene-inactivation strategy to study the gene function, minimize the genome and create pathogen vaccines (Datsenko & Wanner, 2000; Posfai et al., 2006; Ranallo et al., 2006; van Kessel et al., 2008; Gerlach et al., 2009; Katashkina et al., 2009). Three recombineering systems differentiated by the existence status of λ Red genes are filipin available in E. coli. The first is the plasmid-based system, with pKD46 (Datsenko & Wanner, 2000) and pSC101-BAD-gbaA (Wang et al., 2006) the most often used plasmids. λ Red genes in the plasmids are cloned under promoter pBAD, which is tightly regulated by the l-arabinose-induced expression of transcriptional activator AraC (Guzman et al., 1995). Both plasmids harbor the temperature-sensitive pSC101 replicon, which should be maintained at 30 °C. DY380 (Yu et al., 2000; Lee et al., 2001) is the strain normally used in the prophage-based system; it was constructed by integrating the λ prophage obtained by deleting some unnecessary genes of λ phage into the E. coli DH10B chromosome. The λ Red genes in DY380 are under the control of the temperature-sensitive pL promoter, which is blocked by the CI857 repressor at 32 °C.