Modular Engineering of Bacterial Two-Component Systems to Encode Novel Signaling Properties

Humans are populated by trillions of bacteria that sense and respond to host-, microbe-, and diet- derived molecules. A major goal of synthetic biology is to engineer microbes with custom sense-respond behaviors for therapeutic and diagnostic applications. Two-component systems (TCSs) are the primary means by which bacteria sense the environment. In a canonical TCS, a histidine kinase phosphorylates its cognate response regulator, eliciting a transcriptional response. However, natural TCSs contain specific steady-state and dynamical input/output (I/O) properties that have evolved to permit the organism to best adapt to different environmental signals. To leverage and multiplex the tens of thousands of TCSs known from sequence for useful applications, a generalizable strategy for designing desired TCS signaling properties is needed. 

Here, we exploit the modular protein domain architecture of TCSs to program diverse signaling properties. Using our previous E. coli light-switchable TCSs, we identify domains and conserved mutations to create new signaling modules that can increase or decrease pathway activity, and show that this approach is generalizable across different TCSs. Furthermore, using these elements we engineer simple feedback and feedforward loops to encode ultrasensitivity, acceleration, pulsing, delay, and memory. We are applying this framework to engineer probiotic bacteria that can sense and remember exposure to diagnostic profiles of disease-associated human gut metabolites and respond with therapeutic molecules.