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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, Richard M. Murray
doi: https://doi.org/10.1101/015438
Emmanuel L. C. de los Santos
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
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Joseph T. Meyerowitz
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
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Stephen L. Mayo
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
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Richard M. Murray
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA
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Abstract

The pursuit of circuits and metabolic pathways of increasing complexity and ro-bustness 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. 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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Posted April 30, 2015.
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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, Richard M. Murray
bioRxiv 015438; doi: https://doi.org/10.1101/015438
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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, Richard M. Murray
bioRxiv 015438; doi: https://doi.org/10.1101/015438

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