Control ofϕC31 integrase-mediated site-specific recombination by protein trans splicing

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 integrasebased system for rapid regulation of site-specific recombination events in vivo. The ϕC31integrase was split into two extein domains, and intein sequences (Npu DnaE N and Ssp DnaE C ) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in E. coli generated a fully functional ϕC31 integrase. Protein splicing is necessary for recombination activity; no activity was observed when the ϕC31 integrase N-and C-terminal extein domains without the intein sequences were co-expressed, nor when a key intein catalytic residue was mutated. As a proof of principle, we used a bistable switch based on an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system. We used araC and tet inducible promoters to regulate the expression of the two parts of the split recombinase. Inversion of a DNA segment containing a constitutive promoter, catalyzed by trans-spliced integrase, switches between RFP and GFP expression only when both inducible promoters are ON. We used the same split inteins to regulate the reconstitution of a split integrase-RDF fusion that efficiently catalyzed the reverse attR x attL recombination, demonstrating that our split-intein regulated recombination system can function as a reversible AND gate in which the forward reaction is catalyzed by the integrase, and the reverse reaction by the integrase-RDF fusion. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

An integrase toolbox to record gene-expression during plant development

“…In contrast, when the full integrase was expressed under the control of either pUBQ10 or p35S, 75% or more of the seedlings were fully switched. This is consistent with reports that the trans-splicing approach delays the integrase switch in E. coli 53 .…”

“…The first of these tools is the split-intein-integrase system which has been characterized in vitro 53 . Inteins are sequences that trigger autocatalytic splicing, making it possible to reconstitute proteins from fragments expressed from two separate constructs 54 , a technique that has been used previously in plants 55 .…”