Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C.M., Colloms, S. D. and Stark, W. M. (2017) Control of serine integrase recombination directionality by fusion with the directionality factor. Nucleic Acids Research, 45(14), pp. 8635-8645. (doi: 10.1093/nar/gkx567) (PMID:28666339) (PMCID:PMC5737554)
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Abstract
Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle.
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Stark, Professor Marshall and Colloms, Dr Sean and Olorunniji, Dr Femi and McQue, Mrs Arlene and Rosser, Professor Susan |
Authors: | Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C.M., Colloms, S. D., and Stark, W. M. |
College/School: | College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
Journal Name: | Nucleic Acids Research |
Publisher: | Oxford University Press |
ISSN: | 0305-1048 |
ISSN (Online): | 1362-4962 |
Published Online: | 28 June 2017 |
Copyright Holders: | Copyright © 2017 The Authors |
First Published: | First published in Nucleic Acids Research 45(14): 86358645 |
Publisher Policy: | Reproduced under a Creative Commons license |
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