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

Olorunniji, FJ, Merrick, C, Rosser, SJ, Smith, MCM, Stark, WM and Colloms, SD (2017) Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family. In: Site-Specific Recombinases. Springer, pp. 303-323. ISBN 978-1-4939-7167-1

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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.

Item Type: Book Section
Uncontrolled Keywords: 0601 Biochemistry and Cell Biology, 0399 Other Chemical Sciences
Subjects: R Medicine > RM Therapeutics. Pharmacology
Divisions: Pharmacy & Biomolecular Sciences
Publisher: Springer
Related URLs:
Date Deposited: 26 Mar 2020 10:27
Last Modified: 26 Mar 2020 10:27
DOI or Identification number: 10.1007/978-1-4939-7169-5_19
URI: http://researchonline.ljmu.ac.uk/id/eprint/12423

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