TY - JOUR T1 - Engineering Transcriptional Regulator Effector Specificity using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor JF - bioRxiv DO - 10.1101/015438 SP - 015438 AU - Emmanuel L. C. de los Santos AU - Joseph T. Meyerowitz AU - Stephen L. Mayo AU - Richard M. Murray Y1 - 2015/01/01 UR - http://biorxiv.org/content/early/2015/04/30/015438.abstract N2 - 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. ER -