Engineering Transcriptional Regulator Effector Specificity Through Rational Design and Rapid Prototyping

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Emmanuel L.C. de los Santos, Joseph T. Meyerowitz, Stephen L. Mayo, and Richard M. Murray
Presented at Synthetic Biology: Engineering, Evolution and Design (SEED), 14-18 July 2014

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, computational protein design was used to create functional variants of qacR, a tetR family repressor, responsive to a new targeted effector. The modified repressors target vanillin, a growth-inhibiting small molecule found in lignocellulosic hydrolysates and other industrial processes. A computatio ally designed library was screened using an in vitro transcription-translation (TX-TL) system. Leads from the in vitro screen were characterized in vivo. Preliminary results demonstrate dose-dependent regulation of a downstream fluorescent reporter by vanillin. These repressor designs provide a starting point for the evolution of improved variants. We believe this process can serve as a framework for designing new sensors for other target compounds.