Curated Optogenetic Publication Database

Abstract: Cells communicate with each other to jointly regulate cellular processes during cellular differentiation and tissue morphogenesis. This multiscale coordination arises through spatiotemporal activity of morphogens to pattern cell signaling and transcriptional factor activity. This coded information controls cell mechanics, proliferation, and differentiation to shape the growth and morphogenesis of organs. While many of the molecular components and physical interactions have been identified in key model developmental systems, there are still many unresolved questions related to the dynamics involved due to challenges in precisely perturbing and quantitatively measuring signaling dynamics. Recently, a broad range of synthetic optogenetic tools have been developed and employed to quantitatively define relationships between signal transduction and downstream cellular responses. These optogenetic tools can control intracellular activities at the single cell or whole tissue scale to direct subsequent biological processes. In this brief review, we highlight a selected set of studies that develop and implement optogenetic tools to unravel quantitative biophysical mechanisms for tissue growth and morphogenesis across a broad range of biological systems through the manipulation of morphogens, signal transduction cascades, and cell mechanics. More generally, we discuss how optogenetic tools have emerged as a powerful platform for probing and controlling multicellular development.

Abstract: Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.

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: Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision.

Abstract: Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

Abstract: Pseudomonas aeruginosa (P. aeruginosa) infection has become an intractable problem worldwide due to the decreasing efficacy of the mainstay therapy, antibiotic treatment. Hence, exploring new drugs and therapies to address this issue is crucial. Here, we construct a chimeric pyocin (ChPy) to specifically kill P. aeruginosa and engineer a near-infrared (NIR) light-responsive strain to produce and deliver this drug. Our engineered bacterial strain can continuously produce ChPy in the absence of light and release it to kill P. aeruginosa via remotely and precisely controlled bacterial lysis induced by NIR light. We demonstrate that our engineered bacterial strain is effective in P. aeruginosa-infected wound therapy in the mouse model, as it eradicated PAO1 in mouse wounds and shortened the wound healing time. Our work presents a potentially spatiotemporal and noninvasively controlled therapeutic strategy of engineered bacteria for the targeted treatment of P. aeruginosa infections.

Abstract: The increasing integration between biological and digital interfaces has led to heightened interest in utilizing biological materials to store digital data, with the most promising one involving the storage of data within defined sequences of DNA that are created by de novo DNA synthesis. However, there is a lack of methods that can obviate the need for de novo DNA synthesis, which tends to be costly and inefficient. Here, in this work, we detail a method of capturing 2-dimensional light patterns into DNA, by utilizing optogenetic circuits to record light exposure into DNA, encoding spatial locations with barcoding, and retrieving stored images via high-throughput next-generation sequencing. We demonstrate the encoding of multiple images into DNA, totaling 1152 bits, selective image retrieval, as well as robustness to drying, heat and UV. We also demonstrate successful multiplexing using multiple wavelengths of light, capturing 2 different images simultaneously using red and blue light. This work thus establishes a 'living digital camera', paving the way towards integrating biological systems with digital devices.

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.

Abstract: Hydrogels with adjustable mechanical properties have been engineered as matrices for mammalian cells and allow the dynamic, mechano-responsive manipulation of cell fate and function. Recent research yields hydrogels, where biological photoreceptors translated optical signals into a reversible and adjustable change in hydrogel mechanics. While their initial application provides important insights into mechanobiology, broader implementation is limited by a small dynamic range of addressable stiffness. Herein, this limitation is overcome by developing a photoreceptor-based hydrogel with reversibly adjustable stiffness from ≈800 Pa to the sol state. The hydrogel is based on star-shaped polyethylene glycol, functionalized with the red/far-red light photoreceptor phytochrome B (PhyB), or phytochrome-interacting factor 6 (PIF6). Upon illumination with red light, PhyB heterodimerizes with PIF6, thus crosslinking the polymers and resulting in gelation. However, upon illumination with far-red light, the proteins dissociate and trigger a complete gel-to-sol transition. The hydrogel's light-responsive mechanical properties are comprehensively characterized and it is applied as a reversible extracellular matrix for the spatiotemporally controlled deposition of mammalian cells within a microfluidic chip. It is anticipated that this technology will open new avenues for the site- and time-specific positioning of cells and will contribute to overcome spatial restrictions.

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: Optogenetics is a technique employing natural or genetically engineered photoreceptors in transgene organisms to manipulate biological activities with light. Light can be turned on or off, and adjusting its intensity and duration allows optogenetic fine-tuning of cellular processes in a noninvasive and spatiotemporally resolved manner. Since the introduction of Channelrhodopsin-2 and phytochrome-based switches nearly 20 years ago, optogenetic tools have been applied in a variety of model organisms with enormous success, but rarely in plants. For a long time, the dependence of plant growth on light and the absence of retinal, the rhodopsin chromophore, prevented the establishment of plant optogenetics until recent progress overcame these difficulties. We summarize the recent results of work in the field to control plant growth and cellular motion via green light-gated ion channels and present successful applications to light-control gene expression with single or combined photoswitches in plants. Furthermore, we highlight the technical requirements and options for future plant optogenetic research.

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: The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

Abstract: Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, e.g., in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in Saccharomyces cerevisiae upon red light stimulation. The split transcription factor is built from the plant-derived optical dimer PhyB and PIF3, which is fused to a DNA-binding domain based on the catalytically dead Cas9 protein (dCas9) and a transactivation domain. This design combines at least two major advantages: first, the sgRNAs, guiding dCas9 to the promoter of interest, can be exchanged in an efficient and straightforward Golden Gate-based cloning approach, which allows for rational or randomized combination of up to four sgRNAs in a single expression array. Second, target gene expression can be rapidly upregulated by short red light pulses in a light dose-dependent manner and returned to the native expression level by applying far-red light without interfering with the cell culture. Using the native yeast gene CYC1 as an example, we demonstrated that PhiReX 2.0 can upregulate CYC1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.

Abstract: Barrier tissues such as the epidermis employ complex signal transduction systems to execute morphogenetic programs and to rapidly respond to environmental cues to promote homeostasis. Recent advances in live-imaging techniques and tools allow precise spatial and temporal monitoring and manipulation of intracellular signaling cascades. Leveraging the chemistry of naturally occurring light-sensitive proteins, genetically encoded fluorescent biosensors have emerged as robust tools for visualizing dynamic signaling events. In contrast, optogenetic protein constructs permit laser-mediated control of signal receptors and effectors within live cells, organoids, and even model organisms. In this paper, we review the basic principles underlying novel biosensors and optogenetic tools and highlight how recent studies in cutaneous biology have leveraged these imaging strategies to illuminate the spatiotemporal signals regulating epidermal development, barrier formation, and tissue homeostasis.

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: The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.

Abstract: Optogenetic techniques have been intensively applied to the nematode Caenorhabditis elegans to investigate its neural functions. However, as most of these optogenetics are responsive to blue light and the animal exhibits avoidance behavior to blue light, the application of optogenetic tools responsive to longer wavelength light has been eagerly anticipated. In this study, we report the implementation in C. elegans of a phytochrome-based optogenetic tool that responds to red/near-infrared light and manipulates cell signaling. We first introduced the SynPCB system, which enabled us to synthesize phycocyanobilin (PCB), a chromophore for phytochrome, and confirmed the biosynthesis of PCB in neurons, muscles, and intestinal cells. We further confirmed that the amount of PCBs synthesized by the SynPCB system was sufficient for photoswitching of phytochrome B (PhyB)-phytochrome interacting factor 3 (PIF3). In addition, optogenetic elevation of intracellular Ca2+ levels in intestinal cells induced a defecation motor program. These SynPCB system and phytochrome-based optogenetic techniques would be of great value in elucidating the molecular mechanisms underlying C. elegans behaviors.

Abstract: Bacteria can be genetically engineered to act as therapeutic delivery vehicles in the treatment of tumors, killing cancer cells or activating the immune system. This is known as bacteria-mediated cancer therapy (BMCT). Tumor invasion, colonization and tumor regression are major biological events, which are directly associated with antitumor effects and are uncontrollable due to the influence of tumor microenvironments during the BMCT process. Here, we developed a genetic circuit for dynamically programming bacterial lifestyles (planktonic, biofilm or lysis), to precisely manipulate the process of bacterial adhesion, colonization and drug release in the BMCT process, via hierarchical modulation of the lighting power density of near-infrared (NIR) light. The deep tissue penetration of NIR offers us a modality for spatio-temporal and non-invasive control of bacterial genetic circuits in vivo. By combining computational modeling with a high-throughput characterization device, we optimized the genetic circuits in engineered bacteria to program the process of bacterial lifestyle transitions by altering the illumination scheme of NIR. Our results showed that programming intratumoral bacterial lifestyle transitions allows precise control of multiple key steps throughout the BMCT process and therapeutic efficacy can be greatly improved by controlling the localization and dosage of therapeutic agents via optimizing the illumination scheme.

Abstract: Secreted reporters have been demonstrated to be simple and useful tools for analyzing transcriptional regulation in mammalian cells. The distinctive feature of these assays is the ability to detect reporter gene expression in the culture supernatant without affecting the cell physiology or leading to cell lysis, which allows repeated experimentation and sampling of the culture medium using the same cell cultures. Secreted embryonic alkaline phosphatase (SEAP) is one of the most widely used reporter, which can be easily detected using colorimetry following incubation with a substrate, such as p-nitrophenol phosphate. In this report, we present detailed procedures for detection and quantification of the SEAP reporter. We believe that this step-by-step protocol can be easily used by researchers to monitor and measure molecular genetic events in a variety of mammalian cells due to its simplicity and ease of handling. Graphical abstract Schematic overview of the workflow described in this protocol.

Abstract: Microbes regulate brain function through the gut-brain axis, deriving the technology to modulate the gut-brain axis in situ by engineered probiotics. Optogenetics offers precise and flexible strategies for controlling the functions of probiotics in situ. However, the poor penetration of most frequently used short wavelength light has limited the application of optogenetic probiotics in the gut. Herein, a red-light optogenetic gut probiotic was applied for drug production and delivery and regulation of the host behaviors. Firstly, a Red-light Optogenetic E. coli Nissle 1917 strain (ROEN) that could respond to red light and release drug product by light-controlled lysis was constructed. The remaining optical power of red light after 3 cm tissue was still able to initiate gene expression of ROEN and produce about approximately 3-fold induction efficiency. To give full play to the in vivo potential of ROEN, its responsive ability of the penetrated red light was tested, and its encapsulation was realized by PH-sensitive alginate microcapsules for further oral administration. The function of ROEN for gut-brain regulation was realized by releasing Exendin-4 fused with anti-neonatal Fc receptor affibody. Neuroprotection and behavioral regulation effects were evaluated in the Parkinson's disease mouse model, after orally administration of ROEN delivering Exendin-4 under optogenetic control in the murine gut. The red-light optogenetic probiotic might be a perspective platform for in situ drug delivery and gut-brain axis regulation.

Abstract: Developing orthogonal chemical communication pathways in diverse synthetic cell communities is a considerable challenge due to the increased crosstalk and interference associated with large numbers of different types of sender-receiver pairs. Herein, the authors control which sender-receiver pairs communicate in a three-membered community of synthetic cells through red and blue light illumination. Semipermeable protein-polymer-based synthetic cells (proteinosomes) with complementary membrane-attached protein adhesion communicate through single-stranded DNA oligomers and synergistically process biochemical information within a community consisting of one sender and two different receiver populations. Different pairs of red and blue light-responsive protein-protein interactions act as membrane adhesion mediators between the sender and receivers such that they self-assemble and socially self-sort into different multicellular structures under red and blue light. Consequently, distinct sender-receiver pairs come into the signaling range depending on the light illumination and are able to communicate specifically without activation of the other receiver population. Overall, this work shows how photoswitchable membrane adhesion gives rise to different self-sorting protocell patterns that mediate member-specific DNA-based communication in ternary populations of synthetic cells and provides a step towards the design of orthogonal chemical communication networks in diverse communities of synthetic cells.

Abstract: The initiation of apical-basal (AB) polarity and the process of mitotic cell division are both characterised by the generation of specialised plasma membrane and cortical domains. These are generated using shared mechanisms, such as asymmetric protein accumulation, Rho GTPase signalling, cytoskeletal reorganisation, vesicle trafficking and asymmetric phosphoinositide distribution. In epithelial tissue, the coordination of AB polarity and mitosis in space and time is important both during initial epithelial development and to maintain tissue integrity and ensure appropriate cell differentiation at later stages. Whilst significant progress has been made in understanding the mechanisms underlying cell division and AB polarity, it has so far been challenging to fully unpick the complex interrelationship between polarity, signalling, morphogenesis, and cell division. However, the recent emergence of optogenetic protein localisation techniques is now allowing researchers to reversibly control protein activation, localisation and signalling with high spatiotemporal resolution. This has the potential to revolutionise our understanding of how subcellular processes such as apical-basal polarity are integrated with cell behaviours such as mitosis and how these processes impact whole tissue morphogenesis. So far, these techniques have been used to investigate processes such as cleavage furrow ingression, mitotic spindle positioning, and in vivo epithelial morphogenesis. This review describes some of the key shared mechanisms of cell division and apical-basal polarity establishment, how they are coordinated during development and how the advance of optogenetic techniques is furthering this research field.

Abstract: Surgical resection is the main treatment option for most solid tumors, yet cancer recurrence after surgical resection remains a significant challenge in cancer therapy. Recent advances in cancer immunotherapy are enabling radical cures for many tumor patients, but these technologies remain challenging to apply because of side effects related to uncontrollable immune system activation. Here, we develop far-red light-controlled immunomodulatory engineered cells (FLICs) that we load into a hydrogel scaffold, enabling the precise optogenetic control of cytokines release (IFN-β, TNF-α, and IL-12) upon illumination. Experiments with a B16F10 melanoma resection mouse model show that FLICs-loaded hydrogel implants placed at the surgical wound site achieve sustainable release of immunomodulatory cytokines, leading to prevention of tumor recurrence and increased animal survival. Moreover, the FLICs-loaded hydrogel implants elicit long-term immunological memory that prevents against tumor recurrence. Our findings illustrate that this optogenetic perioperative immunotherapy with FLICs-loaded hydrogel implants offers a safe treatment option for solid tumors based on activating host innate and adaptive immune systems to inhibit tumor recurrence after surgery. Beyond extending the optogenetics toolbox for immunotherapy, we envision that our optogenetic-controlled living cell factory platform could be deployed for other biomedical contexts requiring precision induction of bio-therapeutic dosage.

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.