TY - JOUR
T1 - Increasing the λ-Red mediated gene deletion efficiency in Escherichia coli using methyl phosphotriester-modified DNA
AU - Chou, Shu Chiao
AU - Lai, Yi Jyun
AU - Zhuo, Xiao Zhen
AU - Chen, Wen Yih
AU - Li, Si Yu
N1 - Publisher Copyright:
© 2022 Taiwan Institute of Chemical Engineers
PY - 2022/8
Y1 - 2022/8
N2 - 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.
AB - 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.
KW - Escherichia coli
KW - Methyl phosphotriester (MPTE)-modified DNA
KW - Single-strand annealing (SSA) model
KW - λ-Red recombineering
UR - http://www.scopus.com/inward/record.url?scp=85127591184&partnerID=8YFLogxK
U2 - 10.1016/j.jtice.2022.104297
DO - 10.1016/j.jtice.2022.104297
M3 - 期刊論文
AN - SCOPUS:85127591184
SN - 1876-1070
VL - 137
JO - Journal of the Taiwan Institute of Chemical Engineers
JF - Journal of the Taiwan Institute of Chemical Engineers
M1 - 104297
ER -