Skip to main content
SpringerLink
Log in

A Simple Method for Introducing Marker-Free Deletions in the Bacillus subtilis Genome

  • Protocol
  • First Online:
Book cover Strain Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 765))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Forster A. C., and Church G. M. (2007) Synthetic biology projects in vitro. Genome Res, 17, 1–6.

    Article  PubMed  CAS  Google Scholar 

  2. 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.

    Article  PubMed  CAS  Google Scholar 

  3. 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.

    Article  PubMed  CAS  Google Scholar 

  4. 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.

    Article  PubMed  CAS  Google Scholar 

  5. 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.

    Article  PubMed  CAS  Google Scholar 

  6. 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.

    Article  PubMed  CAS  Google Scholar 

  7. 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.

    Article  PubMed  CAS  Google Scholar 

  8. 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.

    Article  PubMed  CAS  Google Scholar 

  9. 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.

    Article  PubMed  Google Scholar 

  10. 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.

    Article  PubMed  CAS  Google Scholar 

  11. 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.

    Google Scholar 

  12. Anagnostopoulos C., and Spizizen J. (1961) Requirements for Transformation in Bacillus Subtilis. J Bacteriol, 81, 741–746.

    PubMed  CAS  Google Scholar 

  13. 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.

    PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Graduate School of Information Science, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan

    Takuya Morimoto & Naotake Ogasawara

  2. Biological Science Laboratories, Kao Corporation, Tochigi, Japan

    Katsutoshi Ara & Katsuya Ozaki

Authors
  1. Takuya Morimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katsutoshi Ara
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katsuya Ozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Naotake Ogasawara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Naotake Ogasawara .

Editor information

Editors and Affiliations

  1. Nature Technology Corporation, Innovation Drive 4701, Lincoln, 68521, Nebraska, USA

    James A. Williams

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

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

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-197-0_20

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-196-3

  • Online ISBN: 978-1-61779-197-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation