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Engineering Transcriptional Regulator Effector Specificity using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor

de los Santos, Emmanuel L. C. and Meyerowitz, Joseph T. and Mayo, Stephen L. and Murray, Richard M. (2016) Engineering Transcriptional Regulator Effector Specificity using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor. ACS Synthetic Biology, 5 (4). pp. 287-295. ISSN 2161-5063. http://resolver.caltech.edu/CaltechAUTHORS:20150303-123633257

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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:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1101/015438DOIDiscussion Paper
http://dx.doi.org/10.1021/acssynbio.5b00090DOIArticle
http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00090PublisherArticle
http://pubs.acs.org/doi/suppl/10.1021/acssynbio.5b00090PublisherSupporting Information
ORCID:
AuthorORCID
Murray, Richard M.0000-0002-5785-7481
Additional Information:© 2015 American Chemical Society. Received: May 6, 2015; Publication Date (Web): August 19, 2015. The authors thank Jongmin Kim and Jackson Cahn for reading the manuscript. This research was conducted with support from the Institute for Collaborative Biotechnologies through Grant W911NF-09-0001 from the U.S. Army Research Office. Additional support was granted in part by the Benjamin M. Rosen Bioengineering Center, the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative, and DARPA through the Living Foundries Program. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
Army Research Office (ARO)W911NF-09-0001
Benjamin M. Rosen Bioengineering CenterUNSPECIFIED
Gordon and Betty Moore FoundationGBMF2809
Defense Advanced Research Projects Agency (DARPA)UNSPECIFIED
Subject Keywords:synthetic biology, cell-free systems, in vitro biological circuit prototyping, computational protein design, protein engineering, TX-TL
Record Number:CaltechAUTHORS:20150303-123633257
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20150303-123633257
Official Citation:Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor Emmanuel L. C. de los Santos, Joseph T. Meyerowitz, Stephen L. Mayo, and Richard M. Murray ACS Synthetic Biology 2016 5 (4), 287-295 DOI: 10.1021/acssynbio.5b00090
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:55478
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:03 Mar 2015 21:02
Last Modified:02 Jun 2016 21:05

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