Application of cell-free systems for the design of a glucose-responsive biosensor and for studying the effects of key SARS-CoV-2 proteins

Abstract

Cell-free platforms have been key in the rapid advancement of synthetic biology. They enable the design of metabolic pathways for the production of any desired proteins. Cell-free systems also present many advantages over traditional in vivo systems such as higher synthesis rates, direct manipulation of the chemical environment, and the ability to even produce proteins toxic to cells. In this study, we made use of the PURE cell-free system to construct a genetic circuit to sense glucose by use of a transcription factor, GntR. Towards this aim, we performed tests of its repression and de-repression characteristics on a reporter with a promoter construct containing the GntR operator site. The Pareto Optimal Ensemble Technique was used to determine the parameters of the model for the simulation of this circuit. This work helps to provide a cell-free alternative for rapid point-of-care detection of glucose for diabetics. Additionally, in light of the recent Covid-19 pandemic, we made use of the myTXTL cell-free system to study some key SARS-CoV-2 proteins. We synthesized the viral host translation inhibitor protein, nsp1 in cell-free and tested its effectiveness to inhibit the translation of a reporter in the same cell-free system. Further, we developed a model to simulate the effect of the RNA-dependent RNA Polymerase (RdRp) on viral protein replication in the myTXTL cell-free system. This work paves a way for simulating the effect of RdRp on viral infection in host cells.