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

## TL;DR

Scientists engineered a new sensor for vanillin, a molecule that inhibits cell growth, using computational design and rapid testing in a lab setting.

## Contribution

A new framework for designing and testing molecular sensors using computational design and in vitro screening is introduced.

## Key findings

- Two qacR mutants were developed that respond to vanillin in vitro and in vivo.
- The process combines computational design with rapid in vitro screening to create new regulatory tools.
- The method can be applied to design sensors for other target compounds.

## 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 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.