A novel approach for Escherichia coli genome editing combining in vivo cloning and targeted long-length chromosomal insertion

Despite the abundance of genetic manipulation approaches, particularly for Escherichia coli, new techniques and increased flexibility in the application of existing techniques are required to address novel aims. The most widely used approaches for chromosome editing are based on bacteriophage site-specific and lambda Red/RecET-mediated homologous recombination. In the present study, these techniques were combined to develop a novel approach for in vivo cloning and targeted long-length chromosomal insertion. This approach permits direct lambda Red-mediated cloning of DNA fragment with lengths of 10 kb or greater from the E. coli chromosome into the plasmid vector pGL2, which carries the on of pSC101, the phi 80-attP site of phi 80 phage, and an excisable Cm-R marker bracketed by lambda-attL/attR sites. In pGL2-based recombinant plasmids, the origin of replication can be eliminated in vitro via hydrolysis by Scel endonuclease and recircularization by DNA ligase. The resulting on-less circular recombinant DNA can be used for targeted insertion of the cloned sequence into the chromosome at a selected site via phi 80 phage-specific integrase-mediated recombination using the Dual-In/Out approach (Minaeva et al., 2008). At the final stage of chromosomal editing, the Cm-R-marker can be excised from the chromosome due to expression of the lambda int/xis genes. Notably, the desired fragment can be inserted as multiple copies in the chromosome by combining insertions at different sites in one strain using the P1 general transduction technique (Moore, 2011). The developed approach is useful for the construction of plasmidless, markerless recombinant strains for fundamental and industrial purposes. (C) 2016 The Authors. Published by Elsevier B.V.