Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing
| Autor: | Jane E. Paget, Femi J. Olorunniji, Susan J. Rosser, Arlene L. McPherson, W. Marshall Stark, Makeba Lawson-Williams |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Computational biology
Protein Engineering RS Genome engineering Inteins Substrate Specificity Trans-Splicing QH301 03 medical and health sciences Protein splicing Genetics Escherichia coli Serine Protein Splicing Site-specific recombination Amino Acid Sequence Cloning Molecular QH426 030304 developmental biology Recombination Genetic 0303 health sciences biology Integrases Organisms Genetically Modified 030302 biochemistry & molecular biology Protein engineering Integrase RNA splicing Exteins biology.protein Intein Synthetic Biology and Bioengineering |
| Zdroj: | Nucleic Acids Research Olorunniji, F J, Lawson-Williams, M, Mcpherson, A L, Paget, J E, Stark, W M & Rosser, S J 2019, ' Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing ', Nucleic Acids Research, vol. 47, no. 21, pp. 11452-11460 . https://doi.org/10.1093/nar/gkz936 |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkz936 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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