Engineering an Optogenetic pH-Modulator in Bacteria.

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.