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

Femi J Olorunniji, Makeba Lawson-Williams, Arlene L Mcpherson, Jane E Paget, W Marshall Stark, Susan J Rosser

Research output: Contribution to journalArticlepeer-review

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 C31 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 posttranslational protein trans-splicing in Escherichia coli generated a fully functional C31 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 systemin 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
Original languageEnglish
Pages (from-to)11452-11460
Number of pages9
JournalNucleic Acids Research
Volume47
Issue number21
Early online date31 Oct 2019
DOIs
Publication statusPublished - 2 Dec 2019

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