Curated Optogenetic Publication Database

Abstract: The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation, we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive spit transcription factors in the budding yeast, Saccharomyces cerevisiae. We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering.

Abstract: Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro's components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Abstract: Optogenetic systems use genetically encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light; however, these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to the high-throughput characterization of optogenetic systems across a range of biological systems and applications.

Abstract: Environmental information may be encoded in the temporal dynamics of transcription factor (TF) activation and subsequently decoded by gene promoters to enact stimulus-specific gene expression programs. Previous studies of this behavior focused on the encoding and decoding of information in TF nuclear localization dynamics, yet cells control the activity of TFs in myriad ways, including by regulating their ability to bind DNA. Here, we use light-controlled mutants of the yeast TF Msn2 as a model system to investigate how promoter decoding of TF localization dynamics is affected by changes in the ability of the TF to bind DNA. We find that yeast promoters directly decode the light-controlled localization dynamics of Msn2 and that the effects of changing Msn2 affinity on that decoding behavior are highly promoter dependent, illustrating how cells could regulate TF localization dynamics and DNA binding in concert for improved control of gene expression.

Abstract: Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions. IMPORTANCE Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner. Optogenetically controlled interactions offer advantages over static perturbations or chemically controlled interactions, as light can be manipulated in space and time and does not require the addition of nutrients or antibiotics. Here, we report a system for rapidly achieving light control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase, we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing S. cerevisiae and serves as a proof of principle for implementing this approach in other consortia.

Abstract: Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control integrated with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization mediated by the proteins CRY2 and CIB1. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae. This article is protected by copyright. All rights reserved.

Abstract: Introduction: Controlling gene expression is a fundamental goal of basic and synthetic biology because it allows insight into cellular function and control of cellular activity. We explored the possibility of generating an optogenetic repressor of gene expression in the model organism Saccharomyces cerevisiae by using light to control the nuclear localization of nuclease-dead Cas9, dCas9. Methods: The dCas9 protein acts as a repressor for a gene of interest when localized to the nucleus in the presence of an appropriate guide RNA (sgRNA). We engineered dCas9, the mammalian transcriptional repressor Mxi1, and an optogenetic tool to control nuclear localization (LINuS) as parts in an existing yeast optogenetic toolkit. This allowed expression cassettes containing novel dCas9 repressor configurations and guide RNAs to be rapidly constructed and integrated into yeast. Results: Our library of repressors displays a range of basal repression without the need for inducers or promoter modification. Populations of cells containing these repressors can be combined to generate a heterogeneous population of yeast with a 100-fold expression range. We find that repression can be dialed modestly in a light dose- and intensity-dependent manner. We used this library to repress expression of the lanosterol 14-alpha-demethylase Erg11, generating yeast with a range of sensitivity to the important antifungal drug fluconazole. Conclusions: This toolkit will be useful for spatiotemporal perturbation of gene expression in Saccharomyces cerevisiae. Additionally, we believe that the simplicity of our scheme will allow these repressors to be easily modified to control gene expression in medically relevant fungi, such as pathogenic yeasts.

Abstract: Biological networks sense extracellular stimuli and generate appropriate outputs within the cell that determine cellular response. Biological signal generators are becoming an important tool for understanding how information is transmitted in these networks and controlling network behavior. Signal generators produce well-defined, dynamic, intracellular signals of important network components, such as kinase activity or the concentration of a specific transcription factor. Synthetic biology tools coupled with in silico control have enabled the construction of these sophisticated biological signal generators. Here we review recent advances in biological signal generator construction and their use in systems biology studies. Challenges for constructing signal generators for a wider range of biological networks and generalizing their use are discussed.

Abstract: Perturbations in the concentration of a specific protein are often used to study and control biological networks. The ability to "dial-in" and programmatically control the concentration of a desired protein in cultures of cells would be transformative for applications in research and biotechnology. We developed a culturing apparatus and feedback control scheme which, in combination with an optogenetic system, allows us to generate defined perturbations in the intracellular concentration of a specific protein in microbial cell culture. As light can be easily added and removed, we can control protein concentration in culture more dynamically than would be possible with long-lived chemical inducers. Control of protein concentration is achieved by sampling individual cells from the culture apparatus, imaging and quantifying protein concentration, and adjusting the inducing light appropriately. The culturing apparatus can be operated as a chemostat, allowing us to precisely control microbial growth and providing cell material for downstream assays. We illustrate the potential for this technology by generating fixed and time-varying concentrations of a specific protein in continuous steady-state cultures of the model organism Saccharomyces cerevisiae. We anticipate that this technology will allow for quantitative studies of biological networks as well as external tuning of synthetic gene circuits and bioprocesses.