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Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family

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Site-Specific Recombinases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1642))

Abstract

Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF). Here we present a set of protocols for the overexpression and purification of bacteriophage ϕC31 and Bxb1 integrase and RDF proteins, their use in DNA assembly reactions, and subsequent modification of the resulting DNA assemblies.

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Author information

Authors and Affiliations

  1. Institute of Molecular, Cell and Systems Biology, University of Glasgow, Bower Building, Glasgow, G12 8QQ, Scotland

    Femi J. Olorunniji, W. Marshall Stark & Sean D. Colloms

  2. SynthSys—Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, CH Waddington Building, The King’s Buildings, Mayfield Road, Edinburgh, EH9 3JD, Scotland

    Christine Merrick & Susan J. Rosser

  3. Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK

    Margaret C. M. Smith

Authors
  1. Femi J. Olorunniji
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  2. Christine Merrick
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  3. Susan J. Rosser
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  4. Margaret C. M. Smith
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  5. W. Marshall Stark
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  6. Sean D. Colloms
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Corresponding author

Correspondence to Sean D. Colloms .

Editor information

Editors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Nikolai Eroshenko

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© 2017 Springer Science+Business Media LLC

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Olorunniji, F.J., Merrick, C., Rosser, S.J., Smith, M.C.M., Stark, W.M., Colloms, S.D. (2017). Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family. In: Eroshenko, N. (eds) Site-Specific Recombinases. Methods in Molecular Biology, vol 1642. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7169-5_19

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  • DOI: https://doi.org/10.1007/978-1-4939-7169-5_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7167-1

  • Online ISBN: 978-1-4939-7169-5

  • eBook Packages: Springer Protocols

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