Development and Characterization of a UV-Violet/Green Photoreversible Transcriptional Regulator in E.coli
Doctor of Philosophy
Genetically encoded photoreceptors, or optogenetic tools, have been used to leverage the tractability of light as an input signal to control biological processes in vivo with unrivaled spatiotemporal precision. The Tabor lab has previously engineered green/red and red/far-red photoreversible two-component systems (TCS) for quantitative, programmable control of transcription dynamics. A photoreversible optogenetic tool with spectral sensitivity in the UV-blue region of the visible spectrum would enable new optogenetic applications in single-cell microscopy, and could be combined with existing tools for dynamic control of multiple genes. In this work, we engineer a photoreversible UV-violet/Green transcriptional regulator in E.coli by repurposing the light-switchable cyanobacteriochrome TCS UirS-UirR from Synechocystis PCC 6803. We demonstrate that UirS-UirR regulates the promoter of the low carbon stress-induced small RNA csiR1 (PcsiR1) ~6-fold in response to UV-light. Using a combination of mutations, dose-response experiments, and in vivo phosphorylation measurements, we show that UirS phosphorylates UirR to activate transcription from PcsiR1 in a UV-light dependent manner. This result is in contrast to a proposed sequestration model for UirS-UirR. By measuring the action spectra of UirS-UirR, we show that it responds specifically in the narrow (380-420nm) UV-violet region of the visible spectrum. We show that the gene expression response to input light occurs in minutes and exploit these rapid dynamics to predictably program gene expression signals. By truncating N-terminal protein domains in UirS, we identify an improved sensor with >15-fold light-induced dynamic range but similar response characteristics as UirS. UirS-UirR is the first photoreversible optogenetic tool that responds in the UV-violet region of the visible spectrum, and could be combined with our previously engineered green/red and red/far-red sensors for precise three-input, three-output optogenetic control of transcription, enabling new modes of characterization and control in systems and synthetic biology and metabolic engineering studies. UirS contains a functional ethylene-binding domain similar to the Arabidopsis thaliana ethylene receptors, but is not known have an ethylene response. We have used the E. coli UirS-UirR system for studying the putative ethylene response of UirS heterologously, free of the cross-regulatory networks present in Synechocystis PCC6803. This work provides preliminary evidence that at least in E.coli, UirS does not have a signalling response to ethylene, or that the ethylene response is not transduced through UirR. The UirS-UirR E.coli system provides a test-bed to study the remarkable spectral diversity of the ‘DXCF cyanobacteriochrome’ family of photoreceptors, and the signaling properties of TCSs containing AraC-family DNA binding domains, which are involved in pathogenesis but are poorly understood.