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Engineering transcriptional regulator effector specificity using computational design and in vitrorapid prototyping : developing a vanillin sensor
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Emmanuel de los Santos, L. C., Meyerowitz, Joseph T., Mayo, Stephen L. and Murray, Richard M. (2016) Engineering transcriptional regulator effector specificity using computational design and in vitrorapid prototyping : developing a vanillin sensor. ACS Synthetic Biology, 5 (4). pp. 287-295. doi:10.1021/acssynbio.5b00090 ISSN 2161-5063.
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Official URL: http://dx.doi.org/10.1021/acssynbio.5b00090
Abstract
The pursuit of circuits and metabolic pathways of increasing complexity and robustness in synthetic biology will require engineering new regulatory tools. Feedback control based on relevant molecules, including toxic intermediates and environmental signals, would enable genetic circuits to react appropriately to changing conditions. In this work, variants of qacR, a tetR family repressor, were generated by computational protein design and screened in a cell-free transcription–translation (TX-TL) system for responsiveness to a new targeted effector. The modified repressors target vanillin, a growth-inhibiting small molecule found in lignocellulosic hydrolysates and other industrial processes. Promising candidates from the in vitro screen were further characterized in vitro and in vivo in a gene circuit. The screen yielded two qacR mutants that respond to vanillin both in vitro and in vivo. While the mutants exhibit some toxicity to cells, presumably due to off-target effects, they are prime starting points for directed evolution toward vanillin sensors with the specifications required for use in a dynamic control loop. We believe this process, a combination of the generation of variants coupled with in vitro screening, can serve as a framework for designing new sensors for other target compounds.
| Item Type: | Journal Article | ||||||||
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| Subjects: | Q Science > QH Natural history > QH301 Biology T Technology > TA Engineering (General). Civil engineering (General) |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||
| Library of Congress Subject Headings (LCSH): | Synthetic biology., Biotechnology., Proteins -- Synthesis. | ||||||||
| Journal or Publication Title: | ACS Synthetic Biology | ||||||||
| Publisher: | American Chemical Society | ||||||||
| ISSN: | 2161-5063 | ||||||||
| Official Date: | 15 April 2016 | ||||||||
| Dates: |
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| Volume: | 5 | ||||||||
| Number: | 4 | ||||||||
| Page Range: | pp. 287-295 | ||||||||
| DOI: | 10.1021/acssynbio.5b00090 | ||||||||
| Status: | Peer Reviewed | ||||||||
| Publication Status: | Published | ||||||||
| Access rights to Published version: | Restricted or Subscription Access | ||||||||
| Funder: | Institute for Collaborative Biotechnologies (ICB), Donna and Benjamin M. Rosen Bioengineering Center , Gordon and Betty Moore Foundation (GBMF), California institute of technology | ||||||||
| Grant number: | W911NF-09-0001, GBMF2809 |
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