Abstract
A genetic tool for introducing marker-free deletions is essential for multiple manipulations of genomes. We have developed a simple and efficient method for creating marker-free deletion mutants of Bacillus subtilis through transformation with recombinant PCR products, using the Escherichia coli mazF gene encoding an endoribonuclease that cleaves free mRNAs as a counterselection tool.
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References
Forster A. C., and Church G. M. (2007) Synthetic biology projects in vitro. Genome Res, 17, 1–6.
Kolisnychenko V., Plunkett G., 3 rd, Herring C. D., Feher T., Posfai J., Blattner F. R., and Posfai, G. (2002) Engineering a reduced Escherichia coli genome. Genome Res, 12, 640–647.
Posfai G., Plunkett G., 3 rd, Feher T., Frisch D., Keil G. M., Umenhoffer K., Kolisnychenko V., Stahl B., Sharma S. S., de Arruda M., Burland V., Harcum S. W., and Blattner F. R. (2006) Emergent properties of reduced-genome Escherichia coli. Science, 312, 1044–1046.
Hashimoto M., Ichimura T., Mizoguchi H., Tanaka K., Fujimitsu K., Keyamura K., Ote T., Yamakawa T., Yamazaki Y., Mori H., Katayama T., and Kato J. (2005) Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol Microbiol, 55, 137–149.
Datsenko K. A., and Wanner B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 97, 6640–6645.
Fabret C., Ehrlich S. D., and Noirot P. (2002) A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol, 46, 25–36.
Morimoto T., Kadoya R., Endo K., Tohata M., Sawada K., Liu S., Ozawa T., Kodama T., Kakeshita H., Kageyama Y., Manabe K., Kanaya S., Ara K., Ozaki K., and Ogasawara N. (2008) Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis. DNA Res, 15, 73–81.
Liu S., Endo K., Ara K., Ozaki K., and Ogasawara N. (2008) Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter. Microbiology, 154, 2562–2570.
Zhang X. Z., Yan X., Cui Z. L., Hong Q., and Li S. P. (2006) mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res, 34, e71.
Morimoto T., Ara K., Ozaki K., and Ogasawara N. (2009) A new simple method to introduce marker-free deletions in the Bacillus subtilis genome. Genes Genet Syst, 84, 315–318.
Harwood C. R. and Archibald A. R. (1990) Growth, maintenance and general techniques, in Molecular Biological Methods for Bacillus (John Wiley & Sons, Chichester, New York, Brisbane, Toronto, Singapore), pp. 549.
Anagnostopoulos C., and Spizizen J. (1961) Requirements for Transformation in Bacillus Subtilis. J Bacteriol, 81, 741–746.
Takagi M., Nishioka M., Kakihara H., Kitabayashi M., Inoue H., Kawakami B., Oka M., and Imanaka T. (1997) Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ Microbiol, 63, 4504–4510.
Acknowledgments
We are grateful to Shu Ishikawa for helpful discussions. This work is part of the subproject “Development of a Technology for the Creation of a Host Cell” included within the industrial technology project “Development of Generic Technology for Production Process Starting Productive Function” of the Ministry of Economy, Trade, and Industry, funded by the New Energy and Industrial Technology Development Organization (NEDO), Japan.
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Morimoto, T., Ara, K., Ozaki, K., Ogasawara, N. (2011). A Simple Method for Introducing Marker-Free Deletions in the Bacillus subtilis Genome. In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_20
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DOI: https://doi.org/10.1007/978-1-61779-197-0_20
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