Programming Microbes Using Pulse Width Modulation of Optical Signals

Abstract

Cells transmit and receive information via signalling pathways. A number of studies have revealed that information is encoded in the temporal dynamics of these pathways and has highlighted how pathway architecture can influence the propagation of signals in time and space. The functional properties of pathway architecture can also be exploited by synthetic biologists to enable precise control of cellular physiology. Here, we characterised the response of a bacterial light-responsive, two-component system to oscillating signals of varying frequencies. We found that the system acted as a low-pass filter, able to respond to low-frequency oscillations and unable to respond to high-frequency oscillations. We then demonstrate that the low-pass filtering behavior can be exploited to enable precise control of gene expression using a strategy termed pulse width modulation (PWM). PWM is a common strategy used in electronics for information encoding that converts a series of digital input signals to an analog response. We further show how the PWM strategy extends the utility of bacterial optogenetic control, allowing the fine-tuning of expression levels, programming of temporal dynamics, and control of microbial physiology via manipulation of a metabolic enzyme.

Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth

“…Due to the fact that N -formylmethionine is required to initiate translation of all proteins, changes in MetE abundance should have an immediate impact on methionine availability and, ultimately, protein synthesis. As was recently experimentally verified2435, MetE levels indeed determine the cell growth rate.…”

“…The background fluorescence and maximum induction level vary significantly from day-to-day, as does the dynamical response of the system to step changes in the light input (Supplementary Note 3). The speed of the latter also depends non-trivially on the magnitude and direction of the step9 (a positive-feedback mechanism arising from transcriptional read-through in the CcaS–CcaR plasmid has been suggested as a possible cause for this behaviour24). All these features pose great challenges to the precise dynamic control of sfGFP expression driven by the CcaS–CcaR system, emphasizing the need for closed-loop controllers.…”

“…An open-loop control scheme for MetE had been suggested in the literature24, and we therefore first sought to reconstruct the strain used in that study. Unfortunately, with our experimental setup and growth protocols, the cells failed to grow without methionine, regardless of the light conditions.…”

Optimizing Metabolite Production Using Periodic Oscillations

“…These simple oscillators have led to the development of a fast, robust tunable synthetic oscillator inside living cells [56]. In addition to these oscillators, light inducible systems have shown the potential to modulate gene expression in a highly controllable fashion [58], [59]. While many synthetic oscillatory systems have been rigorously tested computationally and validated experimentally, their incorporation into larger regulatory circuitry has been less explored.…”

How to train your microbe: methods for dynamically characterizing gene networks

“…3a). 96-well plate-based optogenetic instruments have also been built [102] and used to program more basic gene expression dynamics in E. coli and yeast [56,103]. Plate-based devices should be compatible with the method of Olson and coworkers, while increasing throughput relative to the LTA.…”