Post-translational control of genetic circuits using Potyvirus proteases
Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degr...
| Main Authors: | , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
Oxford University Press
2016
|
| Online Access: | http://hdl.handle.net/1721.1/103544 https://orcid.org/0000-0003-0844-4776 https://orcid.org/0000-0002-6469-0229 |
| _version_ | 1826213005979811840 |
|---|---|
| author | Fernandez-Rodriguez, Jesus Voigt, Christopher A. |
| author2 | MIT Synthetic Biology Center |
| author_facet | MIT Synthetic Biology Center Fernandez-Rodriguez, Jesus Voigt, Christopher A. |
| author_sort | Fernandez-Rodriguez, Jesus |
| collection | MIT |
| description | Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits. |
| first_indexed | 2024-09-23T15:41:37Z |
| format | Article |
| id | mit-1721.1/103544 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:41:37Z |
| publishDate | 2016 |
| publisher | Oxford University Press |
| record_format | dspace |
| spelling | mit-1721.1/1035442022-09-29T15:34:45Z Post-translational control of genetic circuits using Potyvirus proteases Fernandez-Rodriguez, Jesus Voigt, Christopher A. MIT Synthetic Biology Center Massachusetts Institute of Technology. Department of Biological Engineering Fernandez-Rodriguez, Jesus Voigt, Christopher A. Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits. National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center, SynBERC EEC0540879) United States. Office of Naval Research (Multidisciplinary University Research Initiative, N00014-11-1-0725) United States. Office of Naval Research (Multidisciplinary University Research Initiative, N00014-13-1-0074) 2016-07-07T20:34:57Z 2016-07-07T20:34:57Z 2016-06 2016-06 Article http://purl.org/eprint/type/JournalArticle 0305-1048 1362-4962 http://hdl.handle.net/1721.1/103544 Fernandez-Rodriguez, Jesus, and Christopher A. Voigt. "Post-translational control of genetic circuits using Potyvirus proteases." Nucleic Acids Research (June 13, 2016), pp.1-10. https://orcid.org/0000-0003-0844-4776 https://orcid.org/0000-0002-6469-0229 en_US http://dx.doi.org/10.1093/nar/gkw537 Nucleic Acids Research Creative Commons Attribution Non-Commercial License http://creativecommons.org/licenses/by-nc/4.0/ application/pdf Oxford University Press Oxford University Press |
| spellingShingle | Fernandez-Rodriguez, Jesus Voigt, Christopher A. Post-translational control of genetic circuits using Potyvirus proteases |
| title | Post-translational control of genetic circuits using Potyvirus proteases |
| title_full | Post-translational control of genetic circuits using Potyvirus proteases |
| title_fullStr | Post-translational control of genetic circuits using Potyvirus proteases |
| title_full_unstemmed | Post-translational control of genetic circuits using Potyvirus proteases |
| title_short | Post-translational control of genetic circuits using Potyvirus proteases |
| title_sort | post translational control of genetic circuits using potyvirus proteases |
| url | http://hdl.handle.net/1721.1/103544 https://orcid.org/0000-0003-0844-4776 https://orcid.org/0000-0002-6469-0229 |
| work_keys_str_mv | AT fernandezrodriguezjesus posttranslationalcontrolofgeneticcircuitsusingpotyvirusproteases AT voigtchristophera posttranslationalcontrolofgeneticcircuitsusingpotyvirusproteases |