P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads
© 2020 The Authors. Published under the terms of the CC BY 4.0 license Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces b...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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EMBO
2021
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| Online Access: | https://hdl.handle.net/1721.1/133599 |
| _version_ | 1826207215405498368 |
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| author | Park, Yongjin Espah Borujeni, Amin Gorochowski, Thomas E Shin, Jonghyeon Voigt, Christopher A |
| author2 | Massachusetts Institute of Technology. Synthetic Biology Center |
| author_facet | Massachusetts Institute of Technology. Synthetic Biology Center Park, Yongjin Espah Borujeni, Amin Gorochowski, Thomas E Shin, Jonghyeon Voigt, Christopher A |
| author_sort | Park, Yongjin |
| collection | MIT |
| description | © 2020 The Authors. Published under the terms of the CC BY 4.0 license Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces burden, but this decreases performance and can interfere with native transcription. We have designed genomic landing pads in Escherichia coli at high-expression sites, flanked by ultrastrong double terminators. DNA payloads >8 kb are targeted to the landing pads using phage integrases. One landing pad is dedicated to carrying a sensor array, and two are used to carry genetic circuits. NOT/NOR gates based on repressors are optimized for the genome and characterized in the landing pads. These data are used, in conjunction with design automation software (Cello 2.0), to design circuits that perform quantitatively as predicted. These circuits require fourfold less RNA polymerase than when carried on a plasmid and are stable for weeks in a recA+ strain without selection. This approach enables the design of synthetic regulatory networks to guide cells in environments or for applications where plasmid use is infeasible. |
| first_indexed | 2024-09-23T13:46:10Z |
| format | Article |
| id | mit-1721.1/133599 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T13:46:10Z |
| publishDate | 2021 |
| publisher | EMBO |
| record_format | dspace |
| spelling | mit-1721.1/1335992023-08-10T20:23:37Z P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads Park, Yongjin Espah Borujeni, Amin Gorochowski, Thomas E Shin, Jonghyeon Voigt, Christopher A Massachusetts Institute of Technology. Synthetic Biology Center Massachusetts Institute of Technology. Department of Biological Engineering © 2020 The Authors. Published under the terms of the CC BY 4.0 license Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces burden, but this decreases performance and can interfere with native transcription. We have designed genomic landing pads in Escherichia coli at high-expression sites, flanked by ultrastrong double terminators. DNA payloads >8 kb are targeted to the landing pads using phage integrases. One landing pad is dedicated to carrying a sensor array, and two are used to carry genetic circuits. NOT/NOR gates based on repressors are optimized for the genome and characterized in the landing pads. These data are used, in conjunction with design automation software (Cello 2.0), to design circuits that perform quantitatively as predicted. These circuits require fourfold less RNA polymerase than when carried on a plasmid and are stable for weeks in a recA+ strain without selection. This approach enables the design of synthetic regulatory networks to guide cells in environments or for applications where plasmid use is infeasible. 2021-10-27T19:53:44Z 2021-10-27T19:53:44Z 2020 2021-09-10T16:50:32Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/133599 en 10.15252/MSB.20209584 Molecular Systems Biology Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf EMBO EMBO Press |
| spellingShingle | Park, Yongjin Espah Borujeni, Amin Gorochowski, Thomas E Shin, Jonghyeon Voigt, Christopher A P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads |
| title | P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads |
| title_full | P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads |
| title_fullStr | P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads |
| title_full_unstemmed | P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads |
| title_short | P recision design of stable genetic circuits carried in highly‐insulated E. coli genomic landing pads |
| title_sort | p recision design of stable genetic circuits carried in highly insulated e coli genomic landing pads |
| url | https://hdl.handle.net/1721.1/133599 |
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