Optogenetic programming of complex, multiplexed gene expression signals
Olson, Evan J
Tabor, Jeffrey J
Doctor of Philosophy
Optogenetic tools are genetically expressed signaling pathways that transduce extracellular light signals into intracellular, biochemical signals. These biological signals can be used to interface with intracellular biological networks, enabling a perturbative experimental approach that can be used to reverse-engineer the molecular basis underlying cellular behaviors. Light is particularly well suited as an experimentally tunable control signal, because the intensity and wavelength of light sources can be exquisitely controlled in both time and space. Many optogenetic tools have been developed in the past decade; however, the ability to use them to perform the biological function generation required for the interrogation of cellular networks has been limited. Here, I have worked to overcome these limitations by 1) establishing the concept of a biological function generator and identifying a roadmap for the optogenetic characterization of biological systems, 2) developing and demonstrating the first biological function generator used to characterize a cellular circuit in live E. coli cells, 3) implementing a photoconversion-based multispectral model which enables gene expression programming with any light input signal, and 4) developing the first multiplexed optogenetic tool capable of simultaneous, independent generation of two biological functions. This work has produced the world's most precise means of producing arbitrary gene expression signals in live cells. The approach and tools developed here should be generalizable to other optogenetic systems, even in eukaryotic organisms. I describe the technical developments in hardware, software, laboratory protocols, and mathematical models which were required to make this progress. The biological function generator approach and tools developed here are an unprecedented means for characterizing biological systems and controlling cellular behaviors, and will enable novel experimental approaches in both systems and synthetic biology.