Real time genetic compensation operationally defines the dynamic demands of feedback control

SUMMARY Biological signaling networks use feedback control to dynamically adjust their operation in real time. Traditional static genetic methods such as gene knockouts or rescue experiments often can identify the existence of feedback interactions, yet are unable to determine what feedback dynamics are required. Here, we implement a new strategy, closed loop optogenetic compensation (CLOC), to address this problem. Using a custom-built hardware and software infrastructure, CLOC monitors in real time the output of a pathway deleted for a feedback regulator. A minimal model uses these measurements to calculate and deliver—on the fly—an optogenetically-enabled transcriptional input designed to compensate for the effects of the feedback deletion. Application of CLOC to the yeast pheromone response pathway revealed surprisingly distinct dynamic requirements for three well-studied feedback regulators. CLOC, a marriage of control theory and traditional genetics, presents a broadly applicable methodology for defining the dynamic function of biological feedback regulators.