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Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

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

Femi J Olorunniji¹, Christine Merrick², Susan J Rosser²

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

Methods in Molecular Biology (Clifton, N.J.)

|August 18, 2017

Abstract

None:

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.

Keywords:

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

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