Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing.
Amino Acid Sequence
Cloning, Molecular
/ methods
Escherichia coli
/ enzymology
Exteins
/ genetics
Integrases
/ metabolism
Inteins
/ genetics
Organisms, Genetically Modified
Protein Engineering
Protein Splicing
/ genetics
Recombination, Genetic
Serine
/ metabolism
Substrate Specificity
/ genetics
Trans-Splicing
/ genetics
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
02 12 2019
02 12 2019
Historique:
accepted:
07
10
2019
revised:
30
09
2019
received:
09
02
2019
pubmed:
2
11
2019
medline:
2
6
2020
entrez:
1
11
2019
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31667500
pii: 5610344
doi: 10.1093/nar/gkz936
pmc: PMC6868429
doi:
Substances chimiques
Serine
452VLY9402
Integrases
EC 2.7.7.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
11452-11460Subventions
Organisme : Medical Research Council
ID : MC_PC_16077
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M018229/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/003356/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M018040/1
Pays : United Kingdom
Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.
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