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Engineering Transcriptional Regulator Effector Specificity Using Computational Design and In Vitro Rapid Prototyping: Developing a Vanillin Sensor. | LitMetric

AI Article Synopsis

  • The study focuses on developing new regulatory tools for synthetic biology to create more complex and resilient genetic circuits that can adapt to changing conditions using feedback control.
  • Researchers engineered variants of the qacR repressor to respond specifically to vanillin, a molecule that inhibits growth and is present in certain industrial processes.
  • Two promising qacR mutants were identified that work effectively in both laboratory and live cell settings, setting the stage for further refinement and development of vanillin sensors in future applications.

Article 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. While the mutants exhibit some toxicity to cells, presumably due to off-target effects, they are prime starting points for directed evolution toward vanillin sensors with the specifications required for use in a dynamic control loop. 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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Source
http://dx.doi.org/10.1021/acssynbio.5b00090DOI Listing

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