1.
Construction and Characterization of Light-Responsive Transcriptional Systems.
Abstract:
Optogenetic tools provide a means for controlling cellular processes that is rapid, noninvasive, and spatially and temporally precise. With the increase in available optogenetic systems, quantitative comparisons of their performances become important to guide experiments. In this chapter, we first discuss how photoreceptors can be repurposed for light-mediated control of transcription. Then, we provide a detailed protocol for characterizing light-regulated transcriptional systems in budding yeast using fluorescence time-lapse microscopy and mathematical modeling, expanding on our recent publication (Gligorovski et al., Nat Commun 14:3810, 2023).
2.
Multidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor.
Abstract:
The ability to independently control the expression of different genes is important for quantitative biology. Using budding yeast, we characterize GAL1pr, GALL, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr, Z3EV, blue-light inducible optogenetic systems El222-LIP, El222-GLIP, and red-light inducible PhyB-PIF3. We report kinetic parameters, noise scaling, impact on growth, and the fundamental leakiness of each system using an intuitive unit, maxGAL1. We uncover disadvantages of widely used tools, e.g., nonmonotonic activity of MET3pr and GALL, slow off kinetics of the doxycycline- and estradiol-inducible systems tetOpr and Z3EV, and high variability of PHO5pr and red-light activated PhyB-PIF3 system. We introduce two previously uncharacterized systems: strongLOV, a more light-sensitive El222 mutant, and ARG3pr, which is induced in the absence of arginine or presence of methionine. To demonstrate fine control over gene circuits, we experimentally tune the time between cell cycle Start and mitosis, artificially simulating near-wild-type timing. All strains, constructs, code, and data ( https://promoter-benchmark.epfl.ch/ ) are made available.
