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ACS Synth Biol. 2018 Jan 19;7(1):16-23. doi: 10.1021/acssynbio.7b00260. Epub 2017 Oct 12.

Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance.

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School of Engineering, University of Vermont , Burlington, Vermont 05405, United States.
Biomedical Engineering, Boston University , Boston, Massachusetts 02215, United States.


Feedback control allows cells to dynamically sense and respond to environmental changes. However, synthetic controller designs can be challenging because of implementation issues, such as determining optimal expression levels for circuit components within a feedback loop. Here, we addressed this by coupling rational design with selection to engineer a synthetic feedback circuit to optimize tolerance of Escherichia coli to the biojet fuel pinene. E. coli can be engineered to produce pinene, but it is toxic to cells. Efflux pumps, such as the AcrAB-TolC pump, can improve tolerance, but pump expression impacts growth. To address this, we used feedback to dynamically regulate pump expression in response to stress. We developed a library with thousands of synthetic circuit variants and subjected it to three types of pinene treatment (none, constant, and varying pinene). We were able to select for strains that were biofuel tolerant without a significant growth cost in the absence of biofuel. Using next-generation sequencing, we found common characteristics in the designs and identified controllers that dramatically improved biofuel tolerance.


biofuel tolerance; efflux pump; feedback control; next generation sequencing

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