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Published April 15, 2016 | Supplemental Material + Submitted
Journal Article Open

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


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.

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.

Attached Files

Submitted - 015438.full.pdf

Supplemental Material - sb5b00090_si_001.pdf

Supplemental Material - sb5b00090_si_002.zip


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August 20, 2023
October 23, 2023