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

  • Femi J. Olorunniji
  • Christine Merrick
  • Susan J. Rosser
  • Margaret C. M. Smith
  • W. Marshall Stark
  • Sean D. CollomsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1642)


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.

Key words

Site-specific recombination DNA assembly Large serine integrase Bacteriophage ϕC31 Synthetic biology Metabolic engineering 


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Femi J. Olorunniji
    • 1
  • Christine Merrick
    • 2
  • Susan J. Rosser
    • 2
  • Margaret C. M. Smith
    • 3
  • W. Marshall Stark
    • 1
  • Sean D. Colloms
    • 1
    Email author
  1. 1.Institute of Molecular, Cell and Systems Biology, University of GlasgowGlasgowScotland
  2. 2.SynthSys—Synthetic and Systems Biology, School of Biological Sciences, University of EdinburghEdinburghScotland
  3. 3.Department of BiologyUniversity of YorkYorkUK

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