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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Olorunniji FJ,Lawson-Williams M,McPherson AL,Paget JE,Stark WM,Rosser SJdoi
10.1093/nar/gkz936subject
Has Abstractpub_date
2019-12-02 00:00:00pages
11452-11460issue
21eissn
0305-1048issn
1362-4962pii
5610344journal_volume
47pub_type
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