Curated Optogenetic Publication Database

Abstract: Cells in many naturally occurring organisms routinely cooperate to control their extracellular pH in a dynamic and reversible manner, but this capability has been underexplored in synthetic biology. Here, we sought to engineer a microbial system that switches between two states -high and low extracellular pH- with minimal human intervention. We accomplished this by combining: (1) a genetic circuit that produces recombinant urease under the control of a light-inducible promoter; (2) a degradation tag on urease to accelerate the high-to-low pH transition; and (3) optimization of several environmental factors, including media composition, replenishment rate, and light exposure patterns. The system raises the pH when urease is produced and hydrolyzes urea in the media to produce ammonia; it lowers the pH as a byproduct of the cell's native metabolism when urease production ceases. We demonstrate that the optimized system cycles continuously for up to 14 days with minimal performance loss. Overall, our system demonstrates synthetic pH control in an engineered living system and highlights challenges and potential solutions for using such systems outside of the context of typical laboratory manipulation.

Abstract: Synthetic biology has enabled the creation of biological reconfigurable circuits, which perform multiple functions monopolizing a single biological machine; Such a system can switch between different behaviours in response to environmental cues. Previous work has demonstrated switchable dynamical behaviour employing reconfigurable logic gate genetic networks. Here we describe a computational framework for reconfigurable circuits in E.coli using combinations of logic gates, and also propose the biological implementation. The proposed system is an oscillator that can exhibit tunability of frequency and amplitude of oscillations. Further, the frequency of operation can be changed optogenetically. Insilico analysis revealed that two-component light systems, in response to light within a frequency range, can be used for modulating the frequency of the oscillator or stopping the oscillations altogether. Computational modelling reveals that mixing two colonies of E.coli oscillating at different frequencies generates spatial beat patterns. Further, we show that these oscillations more robustly respond to input perturbations compared to the base oscillator, to which the proposed oscillator is a modification. Compared to the base oscillator, the proposed system shows faster synchronization in a colony of cells for a larger region of the parameter space. Additionally, the proposed oscillator also exhibits lesser synchronization error in the transient period after input perturbations. This provides a strong basis for the construction of synthetic reconfigurable circuits in bacteria and other organisms, which can be scaled up to perform functions in the field of time dependent drug delivery with tunable dosages, and sets the stage for further development of circuits with synchronized population level behaviour.

Abstract: We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS UirS-UirR from Synechocystis PCC6803 to E. coli. We demonstrate that the promoter of the small RNA csiR1, previously shown to be activated by inorganic carbon stress, is a UirS-UirR output. Additionally, in contrast to a recently proposed sequestration model, we show that the sensor histidine kinase UirS phosphorylates the response regulator UirR to activate PcsiR1 transcription in response to UV-violet light. Finally, we measure changes in UirS-UirR output minutes after a change in light input and exploit these rapid dynamics to program a challenging gene expression signal with high predictability. UirS-UirR is the first engineered transcriptional regulatory tool activated exclusively by UV-violet light, and the most blue shifted photoreversible transcriptional regulatory tool.