Abstract
Bacterial transformation is an essential component of many molecular biological techniques, but bacterial restriction-modification (R-M) systems can preclude the efficient introduction of shuttle vector plasmids into target bacterial cells. Whole-genome DNA sequences have recently been published for a variety of bacteria. Using homology and motif analyses, putative R-M genes can be identified from genome sequences. Introducing DNA methyltransferase genes into Escherichia coli cells causes subsequently transformed plasmids to be modified by these enzymes. We propose a new method, designated Plasmid Artificial Modification (PAM). A PAM plasmid encoding the modification enzymes expressed by the target bacterial host is transformed into E. coli (PAM host). Propagation of a shuttle vector from the PAM host to the target bacterium ensures that the plasmid will be modified such that it is protected from restriction endonuclease digestion in the target bacterium. The result will be a higher transformation efficiency. Here, we describe the use of PAM and electroporation to transform Bifidobacterium adolescentis ATCC15703. By introducing two genes encoding modification enzymes, we improved transformation efficiency 105-fold.
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Acknowledgments
The author would like to thank Professor M. Shimizu-Kadota for useful discussion. This work was partly supported by the Grant-in-Aid for Scientific Research on Priority Areas in Applied Genomics from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Suzuki, T., Yasui, K. (2011). Plasmid Artificial Modification: A Novel Method for Efficient DNA Transfer into Bacteria. In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_18
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DOI: https://doi.org/10.1007/978-1-61779-197-0_18
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