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

• DNA intermediate is an identified factor in improving the recombineering efficiency. • A short ssDNA is methyl phosphotriester-modified and termed MPTE DNA. • The use of MPTE DNA increases the recombineering efficiency by an order of magnitude. • MPTE DNA cannot serve as a template in PCR. 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. The methyl phosphotriester (MPTE)-modified DNA (MPTE DNA) was used as a supplemental molecule during the electroporation of linear DNA, where the MPTE DNA 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. This study is the first to demonstrate that MPTE DNAs 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 MPTE DNA acts a protective agent and forms the stable ssDNA- MPTE DNA 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 MPTE DNA. The DNA duplex formation with the DNA protective agent may apply to other genomic or biomedical studies. [Display omitted] [ABSTRACT FROM AUTHOR]