Increasing the λ-Red mediated gene deletion efficiency in Escherichia coli using methyl phosphotriester-modified DNA

Background: Phage λ-Red recombineering is a powerful genetic tool to edit the bacterial genome. With the demand of the multiplex genome editing, the efficiency of recombineering is worthy to be further improved. Methods: The methyl phosphotriester (MPTE)-modified DNA (_MPTEDNA) was used as a supplemental molecule during the electroporation of linear DNA, where the _MPTEDNA was single-stranded, 69-nt long, had 5 methylation sites on its 5′-end region, and had a sequence complementary to the dsDNA for recombineering. Significant findings: This study is the first to demonstrate that _MPTEDNAs enhance the transformation efficiency (TE) for ldhA deletion and for frdABCD deletion by 6- and 12-fold, respectively, with a low total dsDNA loading of 40–70 ng. It is suggested that _MPTEDNA acts a protective agent and forms the stable ssDNA-_MPTEDNA duplex, where the 3′end of ssDNA is critical in both ssDNA-annealing model and recA-dependent double-strand invasion recombination model. When the duplex reaches the target gene site, the ssDNA intermediate is interchanged and annealed due to a higher melting temperature (Tm) between the ssDNA and the target gene site than that between ssDNA and _MPTEDNA. The DNA duplex formation with the DNA protective agent may apply to other genomic or biomedical studies.