We show that difficulties in regulating cellular behavior with synthetic biological circuits may be circumvented using
in silico feedback control. By tracking a circuit's output in Saccharomyces cerevisiae in real time, we precisely control its behavior using an in silico feedback algorithm to compute regulatory inputs implemented through a genetically encoded light-responsive module. Moving control functions outside the cell should enable more sophisticated manipulation of cellular processes whenever real-time measurements of cellular variables are possible.
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At a glance
: Characterization of the light-switched system.
a) Light-switchable gene system based on PhyB-PIF3 interaction. Transformed cells grown in darkness and incubated with the chromophore phycocyanobilin (PCB) synthesize both PhyB(Pr)-GBD and PIF3-GAD fusion proteins. Because PIF3 interacts only with the activated form of PhyB (Pfr), the Gal1 target gene is initially off. Upon exposure to red light, PhyB is rapidly converted into its active Pfr form and binds the PIF3 moiety of PIF3-GAD. The transcription activation domain of Gal4 is therefore recruited to the promoter and induces transcription of the target gene. Exposure to far-red light switches off gene expression by rapidly converting PhyB into its inactive Pr form, causing its dissociation from PIF3-GAD. ( b) The expression of YFP driven by the Gal1 promoter can be repeatedly switched on and off using a train of red (R) and far-red (FR) pulses. The trains of light pulses can serve as a control input, and the amount of YFP plays the role of a controlled output. ( c) Experimental dynamics of cell fluorescence in response to a red pulse followed by a far-red pulse. All pulses have 1-min duration. YFP flow cytometry measurements (squares) were taken every 30 min. Each set of matched colored arrow and output squares and curve represent a distinct experiment in which the far-red input was applied at different times. Spontaneous transition of PhyB Pfr to Pr takes place in the dark (a phenomenon known as 'dark reversion') resulting in dissociation of PIF3 from PhyB. Consequently, cell fluorescence reaches a peak and then declines, as mRNA decays over time. Gray squares and line correspond to a control experiment with chromophore addition and no light exposure. ( d) Simulation of the response to the same input as in c. The model reproduces several essential features of the experimental responses, including peak times and decay dynamics. Slight differences between simulated and experimental responses are due to nonlinear effects and delays that are not captured by the model. ( e) Reversibility of the PhyB-PIF3 interaction. The system does not lose its responsiveness to light over several on-off cycles. ( f) Simulation results for the same input as in e. ( g) Response to multiple red pulses. Multiple applications of red light drive the system to higher expression levels than a single red pulse. ( h) Simulation results for multiple- compared with single-pulse responses.
In silico feedback achieves robust regulation of gene expression fold change.
a) In silico feedback control scheme for the light-activated gene system. ( b) Regulation of average YFP fluorescence to sevenfold over a 7-h period using in silico feedback (orange). A pre-computed light pulse train that achieves set point regulation when applied to the mathematical model (gray) did not achieve the desired fold induction when applied in open loop to the biological construct (green). In contrast, closed-loop feedback control achieves the desired fold induction. OL and CL denotes open- and closed-loop control, respectively. ( c) Regulation of average YFP fluorescence to fourfold above basal over a 7-h period. Open- and closed-loop pulse trains determined as in b. ( d) Regulation of average YFP to fivefold above basal over a 7-h period, starting from a randomly perturbed culture. Closed-loop control achieves the desired set point, irrespective of the initial conditions of the system.