SpringerProtocols: Abstract: Plasmid Artificial Modification: A Novel Method for Efficient DNA Transfer into Bacteria

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 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 (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 Roberts R.J., Vincze T., Posfai J., Macelis D. (2010) REBASE – a database for DNA restriction and modification: enzymes, genes and genomes. Roberts R.J. et al. (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Yasui K., Kano Y., Tanaka K., Watanabe K., Shimizu-Kadota M., Yoshikawa H., Suzuki T. (2009) Improvement of bacterial transformation efficiency using plasmid artificial modification. Elhai J., Vepritskiy A., Muro-Pastor A.M., Flores E., Wolk C.P. (1997) Reduction of conjugal transfer efficiency by three restriction activities of Biswas I., Gruss A., Ehrlich S.D., Maguin E. (1993) High-efficiency gene inactivation and replacement system for gram-positive bacteria. Sugawara H., Ohyama A., Mori H., Kurokawa K. (2009) Microbial Genome Annotation Pipeline (MiGAP) for diverse users. The 20th International Conference on Genome Informatics (GIW2009) Poster and Software Demonstrations (Yokohama), S001-1–2. Guzman L.M., Belin D., Carson M.J., Beckwith J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Mizuguchi H., Nakatsuji M., Fujiwara S., Takagi M., Imanaka T. (1999) Characterization and application to hot start PCR of neutralizing monoclonal antibodies against Zhu B., Cai G., Hall E.O., Freeman G.J. (2007) In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Source.


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