Towards Construction of Programmable Cells to Prevent Infectious Diseases

Signal sensing is critical in bacteria that live in fluctuating environments. However, even a group of isogenic bacteria, subject to identical culture conditions, often exhibit heterogeneity in phenotypes. This multi-stability can be explained by stochastic transitioning among multiple stable states of gene expression which are often the results of positive feedback loops in gene regulatory networks. Although such a "bet-hedging" strategy can increase bacterial fitness in unpredictable environments, such stochastic processes can prevent synthetic biologists from predictably engineering microbial cells that need to face uncontrolled environments. Thus, to fully exploit microbes in synthetic biology applications, complex gene regulatory systems need to be better understood. To this end, we propose to construct "robust" genetic sensors and circuits that endow bacteria with the ability to sense environmental signals (e.g., temperature, light, pH, and oxygen level), perform computational operations, and implement actuations (i.e., generation of toxins to kill parasite eggs).

            By building sensors and circuits from the bottom-up, we have gained design principles for construction of programmable cells. Specifically, we have built robust logic gates that contain modular sensors for temperature or oxygen levels that can be more meaningful signals in the environment than chemical inducers that are used in the lab. Furthermore, we have constructed bistable switches that can generate noise-tolerant responses to fluctuating environmental inputs. These switches are based on the sequestering interaction between a transcription factor (an activator) and an anti-activator along with multiple positive feedback loops. Importantly, these switches are functional over a wide range of parameters and endow the bacteria with a long-lasting memory (~weeks). We are assembling these modular sensors, logic gates, and memory devices to construct robust programmable probiotic strains that can reproduce in the intestine where parasite eggs are produced, and come out of the body with the parasite eggs and kill them. We will present progress towards development of such engineered microbes, which can be programmed to kill parasite eggs only when user-defined conditions are met.