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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.
|Journal||Journal of the Taiwan Institute of Chemical Engineers|
|State||Published - Aug 2022|
- Escherichia coli
- Methyl phosphotriester (MPTE)-modified DNA
- Single-strand annealing (SSA) model
- λ-Red recombineering