期刊
ACS SYNTHETIC BIOLOGY
卷 5, 期 4, 页码 287-295出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.5b00090
关键词
synthetic biology; cell-free systems; in vitro biological circuit prototyping; computational protein design; protein engineering; TX-TL
资金
- Institute for Collaborative Biotechnologies [W911NF-09-0001]
- U.S. Army Research Office
- Benjamin M. Rosen Bioengineering Center
- Gordon and Betty Moore Foundation [GBMF2809]
- DARPA through the Living Foundries Program
- Direct For Computer & Info Scie & Enginr
- Division of Computing and Communication Foundations [1317694] Funding Source: National Science Foundation
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.
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