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Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called Serine Integrase Recombinational Assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterised in vitro.In this report, we compared the activities in vitro of ten orthogonal LSIs, to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in E. coli, using 6 different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up 'poly-part' gene assembly and editing.We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications. This article is protected by copyright. All rights reserved.

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.

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 for rapid regulation of site-specific recombination eventsin vivo.TheφC31integrase was split into two extein domains, and intein sequences(NpuDnaENandSspDnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational proteintrans-splicinginE. coligenerated a fully functionalφC31integrase. Protein splicing is necessary for recombination activity; no activity was observed when theφC31 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 usedaraCandtetinducible promoters to regulate the expression of the two parts of the split recombinase. Inversion of a DNA segment containing a constitutive promoter, catalyzed bytrans-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 reverseattRxattLrecombination, 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.