Improvement of Transformation Efficiency Through In Vitro Methylation and SacⅡ Site Mutation of Plasmid Vector in Bifidobacterium longum MG1
2010
Kim, J.Y., Seoul National University, Seoul, Republic of Korea | Wang, Yan, Seoul National University, Seoul, Republic of Korea | Park, M.S., Anyang Technical College, Anyang, Republic of Korea | Ji, G.E., Seoul National University, Seoul, Republic of Korea
The different cleavage patterns of pYBamy59 plasmid isolated from E. coli DH5α and B. longum MG1 by the cell extract of B. longum MG1 suggested that the main reason for its low transformation efficiency was related to the restriction modification (R-M) system. To confirm the correlation between the R-M system and transformation efficiency, in vitro methylation and site-directed mutagenesis were performed in pYBamy59. Sequence analysis of pYBamy59 fragments digested by the cell extract of B. longum MG1 revealed that all fragments were generated by restriction of the sequence recognized by SacⅡ endonuclease. When pYBamy59 from E. coli was methylated in vitro by CpG or GpC methyltransferase, it was protected from SacⅡ digestion. Site-directed mutagenesis, which removed SacⅡ sites from pYBamy59, or in vitro methylation of pYBamy59 showed 8- to 15-fold increases in the transformation efficiency over intact pYBamy59. Modification of the SacⅡ-related R-M system in B. longum MG1 and in vitro methylation in pYBamy 59 can improve the transformation efficiency in this strain. The results showed that the R-M system is a factor to limit introduction of exogenous DNA, and in vitro modification is a convenient method to overcome the barrier of the R-M system for transformation.
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