(174bw) Electrochemical Control of Gene Expression with a Tunable Redox-Sensitive Transcriptional Regulator

Synthetic biological circuits can be perturbed through thermal, chemical, and optical stimuli to activate/repress protein production or switch between stable states. However, these stimuli can be difficult to control with high spatiotemporal fidelity and/or be difficult to deploy in a therapeutic context. By contrast, the tools of electrochemistry could provide a means for fast, localized, and flexible control of gene expression. Here, we develop a link between applied electrochemical potentials and gene expression. The fumarate and nitrate reductase regulator (FNR) is an oxygen-sensitive, global transcriptional regulator in Escherichia coli. Based on environmental oxygen concentrations, FNR regulates gene expression to facilitate the switch between aerobic and anaerobic metabolic pathways. The sensing mechanism is predominantly electrochemical, such that the redox state of an Fe₄S₄ cluster dictates regulatory activity. We therefore hypothesized that FNR is not only oxygen-sensitive, but it is rather more broadly an electrochemically-sensitive protein.

In this direction, we performed experiments to evaluate the electrochemistry of FNR and its potential for use as a component in engineered gene circuits. We constructed plasmids containing fluorescent protein-encoding genes under redox-sensitive promoters. Specifically, we investigated FNR alongside the superoxide response (SoxR) and the oxidative stress (OxyR) regulators. Using a combination of chemical oxidants and applied electrochemical potentials, we cultured E. coli containing these plasmids under a range of oxidizing and reducing conditions. Overall, we observed that FNR had diverse regulatory capacities, with the ability to up- and down-regulate fluorescent protein expression in response to imposed electrochemical stimuli. Based on our learnings from these experiments, we further investigated the feasibility of constructing gene circuits which confer electrochemically-inducible and electrode-dependent behavior in E. coli. Altogether, this work evidences FNR as a highly tunable regulator that could bridge applied electrochemical potentials and cellular function, enabling the use of electrochemistry to rationally control the behavior of living cells.