Olorunniji, F. J., Merrick, C., Rosser, S. J., Smith, M. C.M., Stark, W. M. and Colloms, S. D. (2017) Multipart DNA assembly using site-specific recombinases from the large serine integrase family. In: Eroshenko, N. (ed.) Site-Specific Recombinases: Methods and Protocols. Series: Methods in molecular biology (1642). Springer New York, pp. 303-323. ISBN 9781493971671 (doi: 10.1007/978-1-4939-7169-5_19)
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Publisher's URL: https://link.springer.com/book/10.1007/978-1-4939-7169-5
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 Sections |
|---|---|
| Keywords: | Bacteriophage ϕC31, DNA assembly, Large serine integrase, Metabolic engineering, Site-specific recombination, Synthetic biology |
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Stark, Professor Marshall and Colloms, Dr Sean and Olorunniji, Dr Femi and Rosser, Professor Susan |
| Authors: | Olorunniji, F. J., Merrick, C., Rosser, S. J., Smith, M. C.M., Stark, W. M., and Colloms, S. D. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
| Journal Name: | Methods in molecular biology (Clifton, N.J.) |
| Publisher: | Springer New York |
| ISSN: | 1940-6029 |
| ISBN: | 9781493971671 |
| Published Online: | 17 August 2017 |
| Related URLs: |
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