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.
Highlights
It has recently become possible to create computational and memory systems in cells [1,2,3] allowing us to foresee many new ways to enhance the applications of living organisms [4,5]
Since the natural DnaE split inteins used in this work require an invariant active site cysteine to remain in the spliced product protein [32,33,34], their use for split integrase reconstitution depends upon the identification of a short region of the protein sequence where insertion of a cysteine does not disrupt activity
We started by analysing the domain structure of serine integrases to determine where to split the protein, since a functional split intein could require introduction of mutations that would remain in the spliced protein product, if suitably placed natural Cys, Ser or Thr residues were unavailable
Summary
It has recently become possible to create computational and memory systems in cells [1,2,3] allowing us to foresee many new ways to enhance the applications of living organisms [4,5]. One group of site-specific recombinases known as the serine integrases is especially suited for the construction of switching devices, because these enzymes promote very efficient and highly directional recombination [11]. For such modules to be useful, fully integrated components of the cell, activity of the recombinase must be tightly regulated, so that switching occurs only when other cellular conditions are fulfilled. We demonstrate a powerful new approach to regulation of serine integrase activity, in which the enzyme itself is assembled by intein-mediated fusion of two precursor components
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
