Skip to main content
SpringerLink
Log in

ET Recombination

DNA Engineering Using Homologous Recombination in E. coli

  • Protocol
Bacterial Artificial Chromosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 256))

Abstract

Recombinogenic engineering, or the modification and cloning of DNA molecules via homologous recombination, has opened up a new era. In contrast to conventional cloning strategies, recombinogenic engineering can be carried out at virtually any chosen position on a given target DNA molecule (which can be small or large). Any type of modification, including sequence deletions, substitutions, and insertions can be carried out via homologous recombination. Because homologous recombination is a precise process of high fidelity, recombinogenic engineering generates DNA clones efficiently and with high precision.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Muyrers, J. P. P., Zhang, Y., and Stewart, A. F. (2001) Recombinogenic engineering: new options for cloning and manipulating DNA. Trends Bioch. Sci. 26, 325–331.

    Article  CAS  Google Scholar 

  2. Zhang, Y., Buchholz, F., Muyrers, J. P. P., and Stewart, A. F. (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nature Genet. 20, 123–128.

    Article  PubMed  CAS  Google Scholar 

  3. Muyrers, J. P. P., Zhang, Y, Testa, G., and Stewart, A. F (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucl. Acids Res. 27, 1555–1557.

    Article  PubMed  CAS  Google Scholar 

  4. Narayanan, K., Williamson, R., Zhang, Y, Stewart, A. F, and Ioannou, P. A. (1999) Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther 6, 442–447.

    Article  PubMed  CAS  Google Scholar 

  5. Yu, D., Ellis, H. M., Lee, E. C, Jenkins, N. A., Copeland, N. G., and Court, D.L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983.

    Article  PubMed  CAS  Google Scholar 

  6. Lee, E. C, Yu, D., Martinez de Velasco, J., et al. (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73(1), 56–65.

    Article  PubMed  CAS  Google Scholar 

  7. Angrand, P. O., Daigle, N., van der Hoeven, F., Scholer, H. R., and Stewart, A. F. (1999) Simplified generation of targeting constructs using ET recombination. Nucl. Acids Res. 27, e16.

    Article  PubMed  CAS  Google Scholar 

  8. 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 

  9. Hill, F., Benes, V., Thomasova, D., Stewart, A. F., Kafatos, F. C, and Ansorge, W. (2000) BAC trimming: minimizing clone overlaps. Genomics 64, 111–113.

    Article  PubMed  CAS  Google Scholar 

  10. Muyrers, J. P. P., Zhang, Y, Buchholz, F, and Stewart, A. F (2000) RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev. 14, 1971–1982.

    PubMed  CAS  Google Scholar 

  11. Muyrers, J. P. P., Zhang, Y, Benes, V., Testa, G., Ansorge, W., and Stewart, A. F (2000) Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Reports 1, 239–243.

    Article  PubMed  CAS  Google Scholar 

  12. Muyrers, J. P. P., Zhang, Y, and Stewart, A. F (2000) ET cloning: Think recombination first. Genetic Engineering, Principles and Methods (Setlow, J. K., ed.), 22, 77–98 Kluwer Academic/Plenum, NY.

    Google Scholar 

  13. Nefedov, M., Williamson, R., and Ioannou, P. A. (2000) Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli. Nucl. Acids Res. 28(17), E79.

    Article  PubMed  CAS  Google Scholar 

  14. Zhang, Y, Muyrers, J. P. P., Testa, G., and Stewart, A. F (2000) DNA cloning by homologous recombination in Escherichia coli. Nature Biotech. 18, 1314–1317.

    Article  CAS  Google Scholar 

  15. Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130.

    PubMed  CAS  Google Scholar 

  16. Sambrook, J. and Russell, D. W (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, Woodbury, NY.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Gene Expression Program, European Molecular Biology Laboratory, Heidelberg, Germany

    Joep P. P. Muyrers

  2. Gene Bridges GmbH, Dresden, Germany

    Youming Zhang & Jeanette M. J. Rientjes

  3. Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany

    Vladmir Benes

  4. BioInnovation Zentrum, University of Technology, Dresden, Germany

    Giuseppe Testa & A. Francis Stewart

  5. c/o Max-Planck Institut for Cell Biology and Genetics, Dresden, Germany

    A. Francis Stewart

Authors
  1. Joep P. P. Muyrers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Youming Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vladmir Benes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Giuseppe Testa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeanette M. J. Rientjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. Francis Stewart
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. The Institute for Genomic Research, Rockville, MD

    Shaying Zhao

  2. Office of Biological Environmental Research, US Department of Energy, Germantown, MD

    Marvin Stodolsky

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Muyrers, J.P.P., Zhang, Y., Benes, V., Testa, G., Rientjes, J.M.J., Stewart, A.F. (2004). ET Recombination. In: Zhao, S., Stodolsky, M. (eds) Bacterial Artificial Chromosomes. Methods in Molecular Biology, vol 256. Humana Press. https://doi.org/10.1385/1-59259-753-X:107

Download citation

  • DOI: https://doi.org/10.1385/1-59259-753-X:107

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-989-6

  • Online ISBN: 978-1-59259-753-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation