Design and application of synthetic gene circuits for measuring intracellular signaling states

Abstract

The recent development of live-cell biosensors has revealed a previously unappreciated world of complex signaling dynamics, both in cultured cells and intact animals. However, these live-cell biosensors are inefficient for large-scale experiments or for in vivo tissues that are inaccessible to live microscopy. To address this problem, the Toettcher Lab recently developed a synthetic gene circuit to detect Erk signaling pulses, called the ‘Recorder of Erk Activity Dynamics’ (READer). My thesis aims to refine and extend the concept of synthetic dynamics-sensitive circuits. First, we model a fully- synthetic version of the READer circuit that does not rely on natural Erk-responsive promoters or transcription factors, and show that it similarly detects pulsatile inputs. Then, we explore additional signal processing functions encoded by the underlying topology of the READer circuit, the type 3 incoherent feedforward loop (FFL), by screening for its ability to perform Boolean function when provided with combinations of inputs. We also similarly investigate Boolean function in other FFL topologies. Finally, we show that combinations of the type 3 incoherent FFL can be linked together to produce a synthetic circuit that detects the phase shift between two pulsatile input signals, such as the out of phase oscillations of Wnt and Notch in the somite clock. Overall, by altering and adding connections in a simple synthetic gene circuit, my thesis produces three synthetic circuits that detect different types of intracellular signaling states. Development of synthetic circuits not only provides insight into how information is encoded in nature, but also allows us to generate new experimental and biological tools.