Design and Implementation of a Glucose-Responsive Genetic Switch Circuit in a Cell Free System
Transcription factors (TF)—proteins that bind to promoter regions in the DNA to either activate or repress transcription—play an important role in biosensing and metabolic engineering applications. Metabolite-responsive TFs have the ability to sense specific metabolites; these small molecules bind to a specific domain on the TF and induce a conformational change of the protein, altering its DNA-binding strength and therefore the transcription rate of the downstream gene. We aim to implement, and further engineer, a prokaryote-derived TF, GntR, that responds to glucose or its derivative, D-gluconate, in a genetic switch circuit in a cell free protein synthesis (CFPS) system. Toward this aim, we constructed an E. coli promoter with the GntR operator site and performed preliminary tests of its repression and de-repression characteristics using CFPS. The CFPS platform has key advantages over in vivo systems, with particular regard to probing the metabolite, transcript, and protein levels due to the absence of a cell wall, and greatly facilitates validation of the metabolic and gene regulatory models we plan to develop for the circuit. Moreover, with recent key improvements to its reaction longevity and protein yield, CFPS is a promising platform for rapid prototyping and implementation of genetic circuits. Taken together, this work provides a framework for further optimization and development of the genetic switch circuit, which can be exploited for therapeutic andbiosynthetic applications.
Cell free biosensor; Cell free protein synthesis; Genetic circuit; Genetic switch; Metabolic engineering; Transcription Factor
Varner, Jeffrey D.
DeLisa, Matthew; Helmann, John D.
M.S., Chemical Engineering
Master of Science
dissertation or thesis