Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing

Olorunniji, F. J., Lawson-Williams, M., McPherson, A. L., Paget, J. E., Stark, W. M. and Rosser, S. J. (2019) Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing. Nucleic Acids Research, 47(21), pp. 11452-11460. (doi: 10.1093/nar/gkz936) (PMID:31667500) (PMCID:PMC6868429)

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Abstract

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

Item Type:Articles
Additional Information:Biotechnology and Biological Sciences Research Council [BB/003356/1, BB/M018040/1, BB/M018229/1]; Medical Research Council Proximity [MC_PC_16077]. Funding for open access charge: Liverpool John Moores University grant number [MC_PC_16077].
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Stark, Professor Marshall and McQue, Mrs Arlene
Authors: Olorunniji, F. J., Lawson-Williams, M., McPherson, A. L., Paget, J. E., Stark, W. M., and Rosser, S. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN:0305-1048
ISSN (Online):1362-4962
Published Online:31 October 2019
Copyright Holders:Copyright © 2019 The Authors
First Published:First published in Nucleic Acids Research 47(21):11452-11460
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
167747A platform for rapid and precise DNA module rearrangements in Synthetic BiologyWilliam StarkBiotechnology and Biological Sciences Research Council (BBSRC)BB/K003356/1MCSB - Molecular Genetics