Curated Optogenetic Publication Database

Abstract: Optogenetics is a powerful approach that enables researchers to use light to dynamically manipulate cellular behavior. Since the first published use of optogenetics in synthetic biology, the field has expanded rapidly, yielding a vast array of tools and applications. Despite its immense potential for achieving high spatiotemporal precision, optogenetics has predominantly been employed as a substitute for conventional chemical inducers. In this short review, we discuss key features of microbial optogenetics and highlight applications for understanding biology, cocultures, bioproduction, biomaterials, and therapeutics, in which optogenetics is more fully utilized to realize goals not previously possible by other methods.

Abstract: Biotechnology offers many opportunities for the sustainable manufacturing of valuable products. The toolbox to optimize bioprocesses includes extracellular process elements such as the bioreactor design and mode of operation, medium formulation, culture conditions, feeding rates, and so on. However, these elements are frequently insufficient for achieving optimal process performance or precise product composition. One can use metabolic and genetic engineering methods for optimization at the intracellular level. Nevertheless, those are often of static nature, failing when applied to dynamic processes or if disturbances occur. Furthermore, many bioprocesses are optimized empirically and implemented with little-to-no feedback control to counteract disturbances. The concept of cybergenetics has opened new possibilities to optimize bioprocesses by enabling online modulation of the gene expression of metabolism-relevant proteins via external inputs (e.g., light intensity in optogenetics). Here, we fuse cybergenetics with model-based optimization and predictive control for optimizing dynamic bioprocesses. To do so, we propose to use dynamic constraint-based models that integrate the dynamics of metabolic reactions, resource allocation, and inducible gene expression. We formulate a model-based optimal control problem to find the optimal process inputs. Furthermore, we propose using model predictive control to address uncertainties via online feedback. We focus on fed-batch processes, where the substrate feeding rate is an additional optimization variable. As a simulation example, we show the optogenetic control of the ATPase enzyme complex for dynamic modulation of enforced ATP wasting to adjust product yield and productivity.

Abstract: Optogenetic actuators have revolutionized the resolution at which biological processes can be controlled. In plants, deployment of optogenetics is challenging due to the need for these light-responsive systems to function in the context of horticultural light environments. Furthermore, many available optogenetic actuators are based on plant photoreceptors that might crosstalk with endogenous signaling processes, while others depend on exogenously supplied cofactors. To overcome such challenges, we have developed Highlighter, a synthetic, light-gated gene expression system tailored for in planta function. Highlighter is based on the photoswitchable CcaS-CcaR system from cyanobacteria and is repurposed for plants as a fully genetically encoded system. Analysis of a re-engineered CcaS in Escherichia coli demonstrated green/red photoswitching with phytochromobilin, a chromophore endogenous to plants, but also revealed a blue light response likely derived from a flavin-binding LOV-like domain. We deployed Highlighter in transiently transformed Nicotiana benthamiana for optogenetic control of fluorescent protein expression. Using light to guide differential fluorescent protein expression in nuclei of neighboring cells, we demonstrate unprecedented spatiotemporal control of target gene expression. We implemented the system to demonstrate optogenetic control over plant immunity and pigment production through modulation of the spectral composition of broadband visible (white) light. Highlighter is a step forward for optogenetics in plants and a technology for high-resolution gene induction that will advance fundamental plant biology and provide new opportunities for crop improvement.

Abstract: Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B12 in the lightactivated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts.

Abstract: Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform – Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially-designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Abstract: The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the optoCRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of optoCRISPRi often exhibit no more than a 10-fold dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light-activated CRISPRi system with a high dynamic range (40 fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, nonessential genes, or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space-time and extensive targets, our study would facilitate further research involving complex gene networks, metabolic flux redirection, or bioprinting.

Abstract: Vitamin B12 (cobalamin) is essential for human health and its deficiency results in anemia and neurological damage. Vitamin B12 exists in different forms with various bioactivity but most sensors are unable to discriminate between them. Here, a whole-cell agglutination assay that is specific for adenosylcobalamin (AboB12), which is one of two bioactive forms, is reported. This biosensor consists of Escherichia coli that express the AdoB12 specific binding domain of CarH at their surface. In the presence of AdoB12, CarH forms tetramers, which leads to specific bacterial cell-cell adhesions and agglutination. These CarH tetramers disassemble upon green light illumination such that reversion of the bacterial aggregation can serve as internal quality control. The agglutination assay has a detection limit of 500 nм AdoB12, works in protein-poor biofluids such as urine, and has high specificity to AdoB12 over other forms of vitamin B12 as also demonstrated with commercially available supplements. This work is a proof of concept for a cheap and easy-to-readout AdoB12 sensor that can be implemented at the point-of-care to monitor high-dose vitamin B12 supplementation.

Abstract: Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye.

Abstract: Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Abstract: Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

Abstract: In recent years, light-responsive systems from the field of optogenetics have been applied to several areas of metabolic engineering with remarkable success. By taking advantage of light's high tunability, reversibility, and orthogonality to host endogenous processes, optogenetic systems have enabled unprecedented dynamical controls of microbial fermentations for chemical production, metabolic flux analysis, and population compositions in co-cultures. In this article, we share our opinions on the current state of this new field of metabolic optogenetics.We make the case that it will continue to impact metabolic engineering in increasingly new directions, with the potential to challenge existing paradigms for metabolic pathway and strain optimization as well as bioreactor operation.

Abstract: Molecular optogenetics is a highly dynamic research field. In the past two years, the field was characterized by the development of new allosteric switches as well as the forward integration of optogenetics research towards application. Further, two areas of research have significantly gathered momentum, the use of optogenetics to control liquid-liquid phase separation as well as the application of optogenetic tools in the extracellular space. Here, we review these areas and discuss future directions.

Abstract: Optogenetic approaches enable light-mediated control of cellular activities using genetically encoded photoreceptors. While optogenetic technology is already well established in neuroscience and fundamental research, the implementation of optogenetic tools in bacteriology is still emerging. Engineered bacteria with the specific optogenetic system that function at the transcriptional or post-translational level can sense and respond to light, allowing optogenetic control of bacterial behaviors. In this review, we give a brief overview of available optogenetic systems, including their mode of action, classification, and engineering strategies, and focus on optogenetic control of bacterial behaviors with the highlight of strategies for use of optogenetic systems.

Abstract: Since the successful introduction of exogenous photosensitive proteins, channelrhodopsin, to neurons, optogenetics has enabled substantial understanding of profound brain function by selectively manipulating neural circuits. In an optogenetic system, optical stimulation can be precisely delivered to brain tissue to achieve regulation of cellular electrical activity with unprecedented spatio-temporal resolution in living organisms. In recent years, the development of various optical actuators and novel light-delivery techniques has greatly expanded the scope of optogenetics, enabling the control of other signal pathways in non-neuronal cells for different biomedical applications, such as phototherapy and immunotherapy. This review focuses on the recent advances in optogenetic regulation of cellular activities for photomedicine. We discuss emerging optogenetic tools and light-delivery platforms, along with a survey of optogenetic execution in mammalian and microbial cells.

Abstract: To understand cell biological processes, like signalling pathways, protein movements, or metabolic processes, precise tools for manipulation are desired. Optogenetics allows to control cellular processes by light and can be applied at a high temporal and spatial resolution. In the last three decades, various optogenetic applications have been developed for animal, fungal, and prokaryotic cells. However, using optogenetics in plants has been difficult due to biological and technical issues, like missing cofactors, the presence of endogenous photoreceptors, or the necessity of light for photosynthesis, which potentially activates optogenetic tools constitutively. Recently developed tools overcome these limitations, making the application of optogenetics feasible also in plants. Here, we highlight the most useful recent applications in plants and give a perspective for future optogenetic approaches in plants science.

Abstract: We review fundamental mechanisms and applications of OptoGels: hydrogels with light-programmable properties endowed by photoswitchable proteins ("optoproteins") found in nature. Light, as the primary source of energy on earth, has driven evolution to develop highly-tuned functionalities, such as phototropism and circadian entrainment. These functions are mediated through a growing family of optoproteins that respond to the entire visible spectrum ranging from ultraviolet to infrared by changing their structure to transmit signals inside of cells. In a recent series of articles, engineers and biochemists have incorporated optoproteins into a variety of extracellular systems, endowing them with photocontrollability. While other routes exist for dynamically controlling material properties, light-sensitive proteins have several distinct advantages, including precise spatiotemporal control, reversibility, substrate selectivity, as well as biodegradability and biocompatibility. Available conjugation chemistries endow OptoGels with a combinatorially large design space determined by the set of optoproteins and polymer networks. These combinations result in a variety of tunable material properties. Despite their potential, relatively little of the OptoGel design space has been explored. Here, we aim to summarize innovations in this emerging field and highlight potential future applications of these next generation materials. OptoGels show great promise in applications ranging from mechanobiology, to 3D cell and organoid engineering, and programmable cell eluting materials.

Abstract: Gene and cell therapies are widely recognized as future cancer therapeutics but poor controllability limits their clinical applications. Optogenetics, the use of light-controlled proteins to precisely spatiotemporally regulate the activity of genes and cells, opens up new possibilities for cancer treatment. Light of specific wavelength can activate the immune response, oncolytic activity and modulate cell signaling in tumor cells non-invasively, in dosed manner, with tissue confined action and without side effects of conventional therapies. Here, we review optogenetic approaches in cancer research, their clinical potential and challenges of incorporating optogenetics in cancer therapy. We critically discuss beneficial combinations of optogenetic technologies with therapeutic nanobodies, T-cell activation and CAR-T cell approaches, genome editors and oncolytic viruses. We consider viral vectors and nanoparticles for delivering optogenetic payloads and activating light to tumors. Finally, we highlight herein the prospects for integrating optogenetics into immunotherapy as a novel, fast, reversible and safe approach to cancer treatment.

Abstract: Harnessing the potential of optogenetics in biology requires methodologies from different disciplines ranging from biology, to mechatronics engineering, to control engineering. Light stimulation of a synthetic optogenetic construct in a given biological species can only be achieved via a suitable light stimulation platform. Emerging optogenetic applications entail a consistent, reproducible, and regulated delivery of light adapted to the application requirement. In this review, we explore the evolution of light-induction hardware-software platforms from simple illumination set-ups to sophisticated microscopy, microtiter plate and bioreactor designs, and discuss their respective advantages and disadvantages. Here, we examine design approaches followed in performing optogenetic experiments spanning different cell types and culture volumes, with induction capabilities ranging from single cell stimulation to entire cell culture illumination. The development of automated measurement and stimulation schemes on these platforms has enabled researchers to implement various in silico feedback control strategies to achieve computer-controlled living systems-a theme we briefly discuss in the last part of this review.

Abstract: Synthetic biology is a field of research in which molecular parts (mostly nucleic acids and proteins) are de novo created or modified and then used either alone or in combination to achieve new functions that can help solve the problems of our modern society. In synthetic microbiology, microbes are employed rather than other organisms or cell-free systems. Optogenetics, a relatively recently established technology that relies on the use of genetically encoded photosensitive proteins to control biological processes with high spatiotemporal precision, offers the possibility to empower synthetic (micro)biology applications due to the many positive features that light has as an external trigger. In this review, we describe recent synthetic microbiology applications that made use of optogenetics after briefly introducing the molecular mechanism behind some of the most employed optogenetic tools. We highlight the power and versatility of this technique, which opens up new horizons for both research and industry.

Abstract: A comprehensive understanding of signaling mechanisms helps interpret fundamental biological processes and restore cell behavior from pathological conditions. Signaling outcome depends not only on the activity of each signaling component but also on their dynamic interaction in time and space, which remains challenging to probe by biochemical and cell-based assays. Opsin-based optogenetics has transformed neural science research with its spatiotemporal modulation of the activity of excitable cells. Motivated by this advantage, opsin-free optogenetics extends the power of light to a larger spectrum of signaling molecules. This review summarizes commonly used opsin-free optogenetic strategies, presents a historical overview of split protein complementation, and highlights the adaptation of split protein recombination as optogenetic sensors and actuators.

Abstract: Unraveling the transformative power of optogenetics in biology requires sophisticated engineering for the creation and optimization of light-regulatable proteins. In addition, diverse strategies have been used for the tuning of these light-sensitive regulators. This review highlights different protein engineering and synthetic biology approaches, which might aid in the development and optimization of novel optogenetic proteins (Opto-proteins). Focusing on non-neuronal optogenetics, chromophore availability, general strategies for creating light-controllable functions, modification of the photosensitive domains and their fusion to effector domains, as well as tuning concepts for Opto-proteins are discussed. Thus, this review shall not serve as an encyclopedic summary of light-sensitive regulators but aims at discussing important aspects for the engineering of light-controllable proteins through selected examples.

Abstract: Engineered, light-sensitive protein switches are used to interrogate a broad variety of biological processes. These switches are typically constructed by genetically fusing naturally occurring light-responsive protein domains with functional domains from other proteins. Protein activity can be controlled using a variety of mechanisms including light-induced colocalization, caging, and allosteric regulation. Protein design efforts have focused on reducing background signaling, maximizing the change in activity upon light stimulation, and perturbing the kinetics of switching. It is common to combine structure-based modeling with experimental screening to identify ideal fusion points between domains and discover point mutations that optimize switching. Here, we introduce commonly used light-sensitive domains and summarize recent progress in using them to regulate protein activity.

Abstract: Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems. Optogenetics technique can be a solution as it not only enables dynamic control with unprecedented spatiotemporal precision but also is inexpensive and eco-friendlier. The optogenetic technique harnesses natural light-sensing modules that are genetically encodable and re-programmable in various hosts. By further engineering these modules to connect with the microbial regulatory machinery, gene expression and protein activity can be finely tuned simply through light irradiation. Recent works on applying optogenetics to microbial synthetic biology have yielded remarkable achievements. To further expand the usability of optogenetics, more optogenetic tools with greater portability that are compatible with different microbial hosts need to be developed. This review focuses on non-opsin optogenetic systems and the current state of optogenetic advancements in microbes, by showcasing the different designs and functions of optogenetic tools, followed by an insight into the optogenetic approaches used to circumvent challenges in synthetic biology.

Abstract: Although the tools based on split proteins have found broad applications, ranging from controlled biological signaling to advanced molecular architectures, many of them suffer from drawbacks such as background reassembly, low thermodynamic stability, and static structural features. Here, we present a chemically inducible protein assembly method enabled by the dissection of the carboxyl-terminal domain of a B12-dependent photoreceptor, CarHC. The resulting segments reassemble efficiently upon addition of cobalamin (AdoB12, MeB12, or CNB12). Photolysis of the cofactors such as AdoB12 and MeB12 further leads to stable protein adducts harboring a bis-His-ligated B12. Split CarHC enables the creation of a series of protein hydrogels, of which the mechanics can be either photostrengthened or photoweakened, depending on the type of B12. These materials are also well suited for three dimensional cell culturing. Together, this new protein chemistry, featuring negligible background autoassembly, stable conjugation, and phototunability, has opened up opportunities for designing smart materials.