Multidimensional single-cell benchmarking of inducible promoters for precise dynamic control in budding yeast

Abstract

For quantitative systems biology, simultaneous readout of multiple cellular processes as well as precise, independent control over different genes activities are essential. In contrast to readout systems such as fluorescent proteins, control systems such as inducible transcription-factor-promoter systems have only been characterized in an ad hoc fashion, impeding precise system-level manipulations of biological systems and reliable modeling. We designed and performed systematic benchmarks involving easy-to-communicate units to characterize and compare inducible transcriptional systems. We built a comprehensive single-copy library of inducible systems controlling standardized fluorescent protein expression in budding yeast, including GAL1pr, GALL, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr, Z3EV system, the blue-light optogenetic systems El222-LIP, El222-GLIP and the red-light inducible PhyB-PIF3 system. To analyze these systems dynamic properties, we performed high-throughput time-lapse microscopy. The analysis of >100 000 cell images was made possible by the recently developed convolutional neural network YeaZ. We report key kinetic parameters, scaling of noise levels, impacts on growth, and, crucially, the fundamental leakiness of each system. Our multidimensional benchmarking additionally uncovers unexpected disadvantages of widely used tools, e.g., nonmonotonic activity of the MET3 and GALL promoters, 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 new tools for controlling gene expression: strongLOV, a more light-sensitive El222 mutant, and ARG3pr that functions as an OR gate induced by the lack of arginine or presence of methionine. To demonstrate the ability to finely control genetic circuits, we experimentally tuned the time between cell cycle Start and mitotic entry in budding yeast, artificially simulating near-wild-type timing. The characterizations presented here define the compromises that need to be made for quantitative experiments in systems and synthetic biology. To calibrate perturbations across laboratories and to allow new inducible systems to be benchmarked, we deposited single-copy reporter yeast strains, plasmids, and computer analysis code in public repositories. Furthermore, this resource can be accessed and expanded through the website https://promoter-benchmark.epfl.ch/.