sb7b00260_si_001.pdf (450.94 kB)
Design and Selection of a Synthetic Feedback Loop for Optimizing Biofuel Tolerance
journal contribution
posted on 2017-10-12, 00:00 authored by Yik Siu, Jesse Fenno, Jessica M. Lindle, Mary J. DunlopFeedback
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.
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circuit variantsE . coliEscherichia coliimplementation issuesbiofuel toleranceexpression impacts growthgrowth costbiojet fuel pinenefeedback circuitOptimizing Biofuel Tolerance Feedback controlSynthetic Feedback Loopfeedback loopEfflux pumpsnext-generation sequencingcircuit componentscontroller designspinene treatmentexpression levels. coli
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