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

BioRxiv : the Preprint Server for Biology
Emmanuel Lorenzo Cornejo de los SantosRichard M Murray

Abstract

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 compu- tational 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.

Related Concepts

Environment
Repressor Proteins
Testolactone
Transcription, Genetic
lignocellulose
Vanillin
Gene Mutant
Transcriptional Regulation
Solocyte
Small Molecule

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