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

Research output: Chapter in Book/Report/Conference proceedingChapter

Published copy (DOI)

Author(s)

  • Femi J Olorunniji
  • Christine Merrick
  • Susan J Rosser
  • Margaret C M Smith
  • W Marshall Stark
  • Sean D Colloms

Department/unit(s)

Publication details

Title of host publicationSite-Specific Recombinases
DatePublished - 2017
Pages303-323
Number of pages21
PublisherHumana Press
EditorsNikolai Eroshenko
Volume1642
Original languageEnglish
ISBN (Electronic)978-1-4939-7169-5
ISBN (Print)978-1-4939-7167-1

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1642
ISSN (Print)1064-3745

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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