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Nucleic Acids Research Advance Access published online on November 12, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn884
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods online

Improvement of bacterial transformation efficiency using plasmid artificial modification

Kazumasa Yasui1, Yasunobu Kano2, Kaori Tanaka3, Kunitomo Watanabe3, Mariko Shimizu-Kadota4,5, Hirofumi Yoshikawa5 and Tohru Suzuki1,*

1The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, 2Department of Molecular Genetics, Kyoto Pharmaceutical University, 1 Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, 3Division of Anaerobe Research, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, 4Department of Environmental Science, Musashino University, Shinmachi Nishitokyo-shi, Tokyo 202-8585 and 5Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

*To whom correspondence should be addressed. Tel: +81 58 293 2996; Fax: +81 58 293 2992; Email: suzuki{at}gifu-u.ac.jp

Received July 22, 2008. Revised October 11, 2008. Accepted October 20, 2008.

We have developed a method to improve the transformation efficiency in genome-sequenced bacteria, using ‘Plasmid Artificial Modification’ (PAM), using the host's own restriction system. In this method, a shuttle vector was pre-methylated in Escherichia coli cells, which carry all the putative genes encoding the DNA modification enzymes of the target microorganism, before electroporation was performed. In the case of Bifidobacterium adolescentis ATCC15703 and pKKT427 (3.9 kb E. coli-Bifidobacterium shuttle vector), introducing two Type II DNA methyltransferase genes lead to an enhancement in the transformation efficiency by five orders of magnitude. This concept was also applicable to a Type I restriction system. In the case of Lactococcus lactis IO-1, by using PAM with a putative Type I methyltransferase system, hsdMS1, the transformation efficiency was improved by a factor of seven over that without PAM.


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