Curated Optogenetic Publication Database

Abstract: Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Tailoring their properties enables their full exploitation for optogenetic regulation in basic research and applied bioengineering applications. Here, we present a simple, yet powerful strategy based on random mutagenesis coupled to high-throughput screening that allowed altering the most fundamental properties of the widely used nMag/pMag photodimerization system: its light sensitivity and activation. Variants were characterized in vivo in bacteria by flow cytometry and during the entire growth curve by spectrofluorometry. We identify mutations that either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the light activation and dark-to-light fold change. Notably, light sensitivity and activation levels could be changed independently. In addition, we demonstrated that the shapes of the dose-response curves can be finely tuned. This broadens the applicability of the Magnets photosensors for optogenetic regulation strategies.

Abstract: Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to control gene expression independently from mammalian transcriptional and/or translational machineries thus working orthogonally to endogenous regulatory mechanisms. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize, and optimize a new optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool is orthogonal to the cellular machinery for transcription and consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7-mOptoT7). In our study we exploited the T7 polymerase's viral origins to tune our system's expression level, reaching up to an almost 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate the gene expression burden when compared to another optogenetic construct. These properties make mOptoT7 a powerful new tool to use when orthogonality and viral RNA species (that lack endogenous RNA modifications) are desired.