Curated Optogenetic Publication Database

Abstract: Photosynthetic cyanobacteria can be utilised in biotechnology as environmentally sustainable cell factories to convert CO2 into a diverse range of biochemicals. However, a lack of molecular tools available for precise and dynamic control of gene expression hinders metabolic engineering and contributes to low product titres. Optogenetic tools enable light-regulated control of gene expression with high tunability and reversibility. To date, their application in cyanobacteria is limited and transferability between species remains unclear. In this study, we expressed the blue light-repressible YF1/FixJ and the green/red light-responsive CcaS/CcaR systems in Synechococcus sp. PCC 7002 and characterised their performance using GFP fluorescence assays and qRT-PCR. The YF1/FixJ system of non-cyanobacterial origin showed poor performance with a maximum dynamic range of 1.5-fold despite several steps to improve this. By contrast, the CcaS/CcaR system originating from the cyanobacterium Synechocystis sp. PCC 6803 responded well to light wavelengths and intensities, with a 6-fold increased protein fluorescence output observed after 30 min of green light. Monitoring GFP transcript levels allowed us to quantify the kinetics of transcriptional activation and deactivation and to test the effect of both multiple green/red and light/dark cycles on system performance. Finally, we increased CcaS/CcaR system activity under green light through targeted genetic modifications to the pCpcG2 output promoter. This study provides a detailed characterisation of the behaviour of the CcaS/CcaR system in Synechococcus sp. PCC 7002, as well as underlining the complexity of transferring optogenetic tools across species.

Abstract: This volume explores the latest advancements in the field of optogenetics and how it uses cellular light-sensing components and genetic engineering to control proteins and biological processes. The book chapters are organized into four parts. Part One focuses on intracellular optogenetic components for control of specific cell functions; Part Two looks at externally supplied light regulators that do not require genetic manipulation of target cells; Part Three highlights optogenetic control of organelles, and Part Four introduces technical tools required for light induction in optogenetic experiments, as well as a method for performing and analyzing optogenetic cell-cell adhesion. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and practical, Optogenetics: Methods and Protocols is a valuable resource to help researchers understand and apply the concepts of optogenetics and the underlying bioengineering principles, and establish the required technical light-illumination setups for administering light inputs and analysis of experimental outcomes.

Abstract: Light as an environmental signal can effectively regulate various biological processes in microbial systems. Optical and optogenetic tools are able to utilize light for precise control methods with minimal interference. Recently, research on these tools has extended to the field of microbiology. Distinguishing from existing reviews, this review narrows the scope of application into food sector, focusing on advances in optical and optogenetic tools for microbial control, including optical tools targeting pathogenic or probiotic bacteria for non-thermal sterilization, antimicrobial photodynamic therapy, or photobiomodulation, combined with nanomaterials as photosensors for food analysis. As well as using optogenetic tools for more convenient and precise control in food production processes, covering reversible induction, metabolic flux regulation, biofilm formation, and inhibition. These tools offer new solutions to goals that cannot be achieved by traditional methods, and they are still maturing to explore other uses in the food field.

Abstract: Research for biopharmaceutical production processes with mammalian cells steadily aims to enhance the cell-specific productivity as a means for optimizing total productivities of bioreactors. Whereas current technologies such as pH, temperature, and osmolality shift require modifications of the cultivation medium, the use of optogenetic switches in recombinant producer cells might be a promising contact-free alternative. However, the proper application of optogenetically engineered cells requires a detailed understanding of basic cellular responses of cells that do not yet contain the optogenetic switches. The knowhow of ideal light exposure to enable the optimum use of related approaches is missing so far. Consequently, the current study set out to find optimum conditions for IgG1 producing Chinese hamster ovary (CHO) cells which were exposed to blue LED light. Growth characteristics, cell-specific productivity using enzyme-linked immunosorbent assay, as well as cell cycle distribution using flow cytometry were analyzed. Whereas too harsh light exposure causes detrimental growth effects that could be compensated with antioxidants, a surprising boost of cell-specific productivity by 57% occurred at optimum high light doses. The increase coincided with an increased number of cells in the G1 phase of the cell cycle after 72 h of illumination. The results present a promising new approach to boost biopharmaceutical productivity of mammalian cells simply by proper light exposure without any further optogenetic engineering. KEY POINTS: • Blue LED light hinders growth in CHO DP-12 cells • Antioxidants protect to a certain degree from blue light effects • Illumination with blue LED light raises cell-specific productivity.

Abstract: Light-Oxygen-Voltage (LOV) domains are the protein-based light switches used in nature to trigger and regulate various processes. They allow light signals to be converted into metabolic signaling cascades. Various LOV-domain proteins have been characterized in the last few decades and have been used to develop light-sensitive tools in cell biology research. LOV-based applications exploit the light-driven regulation of effector elements to activate signaling pathways, activate genes, or locate proteins within cells. A relatively new application of an engineered small LOV-domain protein called miniSOG (mini singlet oxygen generator) is based on the light-induced formation of reactive oxygen species (ROS). The first miniSOG was engineered from a LOV domain from Arabidopsis thaliana. This engineered 14 kDa light-responsive flavin-containing protein can be exploited as protein tag for the light-triggered localized production of ROS. Such tunable ROS production by miniSOG or similarly redesigned LOV-domains can be of use in studies focused on subcellular phenomena but may also allow new light-fueled catalytic processes. This review provides an overview of the discovery of LOV domains and their development into tools for cell biology. It also highlights recent advancements in engineering LOV domains for various biotechnological applications and cell biology studies.

Abstract: Spatial organization of microbes in biofilms enables crucial community function such as division of labor. However, quantitative understanding of such emergent community properties remains limited due to a scarcity of tools for patterning heterogeneous biofilms. Here we develop a synthetic optogenetic toolkit 'Multipattern Biofilm Lithography' for rational engineering and orthogonal patterning of multi-strain biofilms, inspired by successive adhesion and phenotypic differentiation in natural biofilms. We apply this toolkit to profile the growth dynamics of heterogeneous biofilm communities, and observe the emergence of spatially modulated commensal relationships due to shared antibiotic protection against the beta-lactam ampicillin. Supported by biophysical modeling, these results yield in-vivo measurements of key parameters, e.g., molecular beta-lactamase production per cell and length scale of antibiotic zone of protection. Our toolbox and associated findings provide quantitative insights into the spatial organization and distributed antibiotic protection within biofilms, with direct implications for future biofilm research and engineering.

Abstract: The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis of action spectra. Current methods to determine action spectra only allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination. The RainbowCap provides equal photon flux for each wavelength, with the output of the LEDs narrowed by optical bandpass filters. We validated the RainbowCap for photoactivatable G protein-coupled receptors (opto-GPCRs) and enzymes for the control of intracellular downstream signaling. The simultaneous, spectrally defined illumination provides minimal interruption during time-series measurements, while resolving 10 nm differences in the action spectra of optogenetic proteins under identical experimental conditions. The RainbowCap is also suitable for studying the spectral dependence of light-regulated gene expression in bacteria, which requires illumination over several hours. In summary, the RainbowCap provides high-throughput spectral illumination of microplates, while its modular, customizable design allows easy adaptation to a wide range of optogenetic and photobiological applications.

Abstract: Oscillations are a recurrent phenomenon in biological systems across scales, but deciphering their fundamental principles is very challenging. Here, we tackle this challenge by redesigning the wellcharacterised synthetic oscillator known as “repressilator” in Escherichia coli and controlling it using optogenetics, creating the “optoscillator”. Bacterial colonies manifest oscillations as spatial ring patterns. When we apply periodic light pulses, the optoscillator behaves as a forced oscillator and we systematically investigate the properties of the rings under various light conditions. Combining experiments with mathematical modeling, we demonstrate that this simple oscillatory circuit can generate complex dynamics that are transformed into distinct spatial patterns. We report the observation of synchronisation, resonance, subharmonic resonance and period doubling. Furthermore, we present evidence of a chaotic regime. This work highlights the intricate spatiotemporal patterns accessible by synthetic oscillators and underscores the potential of our approach in revealing fundamental principles of biological oscillations.

Abstract: Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Abstract: Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over the last two years, there have been notable advances in the development of novel optogenetic switches, their utilization in elucidating intricate signaling pathways, and their progress toward practical applications in biotechnological processes, material sciences, and therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, and contemplate future directions.

Abstract: [This corrects the article DOI: 10.1002/mco2.226.].

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: The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.

Abstract: The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies.

Abstract: Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.

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: Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 μm) and does not fully exploit the features of the "high spatial resolution" of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII.

Abstract: Regenerative medicine aims to restore damaged cells, tissues, and organs, for which growth factors are vital to stimulate regenerative cellular transformations. Major advances have been made in growth factor engineering and delivery like the development of robust peptidomimetics and controlled release matrices. However, their clinical applicability remains limited due to their poor stability in the body and need for careful regulation of their local concentration to avoid unwanted side-effects. In this study, a strategy to overcome these limitations is explored using engineered living materials (ELMs), which contain live microorganisms that can be programmed with stimuli-responsive functionalities. Specifically, the development of an ELM that releases a pro-angiogenic protein in a light-regulated manner is described. This is achieved by optogenetically engineering bacteria to synthesize and secrete a vascular endothelial growth factor peptidomimetic (QK) linked to a collagen-binding domain. The bacteria are securely encapsulated in bilayer hydrogel constructs that support bacterial functionality but prevent their escape from the ELM. In situ control over the release profiles of the pro-angiogenic protein using light is demonstrated. Finally, it is shown that the released protein is able to bind collagen and promote angiogenic network formation among vascular endothelial cells, indicating the regenerative potential of these ELMs.

Abstract: Photoreceptor proteins are versatile toolbox for developing biosensors for optogenetic applications. These molecular tools get activated upon illumination of blue light, which in turn offers a non-invasive method for gaining high spatiotemporal resolution and precise control of cellular signal transduction. The Light-Oxygen-Voltage (LOV) domain family of proteins is a well-recognized system for constructing optogenetic devices. Translation of these proteins into efficient cellular sensors is possible by tuning their photochemistry lifetime. However, the bottleneck is the need for more understanding of the relationship between the protein environment and photocycle kinetics. Significantly, the effect of the local environment also modulates the electronic structure of chromophore, which perturbs the electrostatic and hydrophobic interaction within the binding site. This work highlights the critical factors hidden in the protein networks, linking with their experimental photocycle kinetics. It presents an opportunity to quantitatively examine the alternation in chromophore's equilibrium geometry and identify details which have substantial implications in designing synthetic LOV constructs with desirable photocycle efficiency.

Abstract: Genetically engineered bacteria have aroused attention as micro-nano drug delivery systems in situ. However, conventional designs of engineered bacteria usually function constantly or autonomously, which might be non-specific or imprecise. Therefore, designing and optimizing in situ control strategy are important methodological progress for therapeutic researches of intestinal engineered bacteria. Here, a micro-nano optogenetic system based on probiotic was developed combining microelectronics, nanotechnology, and synthetic biology to achieve in situ controllable drug delivery. Firstly, optogenetic engineered Lactococcus lactis was orally administrated in the intestinal tract. A wearable optical device was designed to control optical signals remotely. Then, L. lactis could be customized to secrete peptides according to optical signals. As an example, optogenetic L. lactis system can be constructed to secrete glucagon-like peptide-1 (GLP-1) under the control of the wearable optical device to regulate metabolism. To improve the half-life of GLP-1 in vivo, Fc-domain fused GLP-1 was optimally used. Using this strategy, blood glucose, weight, and other features were well controlled in rats and mice models. Furthermore, upconversion microcapsules were introduced to increase the excitation wavelength of the optogenetic system for better penetrability. This strategy has biomedical potential to expand the toolbox for intestinal engineered bacteria.

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: The expression of genes of interest (GOI) can be initiated by providing external stimuli such as temperature shifts and light irradiation. The application of thermal or light stimuli triggers structural changes in stimuli-sensitive biomolecules within the cell, thereby inducing or repressing gene expression. Over the past two decades, several groups have reported genetic circuits that use natural or engineered stimuli-sensitive modules to manipulate gene expression. Here, we summarize versatile strategies of thermosensors and light-driven systems for the conditional expression of GOI in bacterial hosts.

Abstract: Engineering bacteria can achieve targeted and controllable cancer therapy using synthetic biology technology and the characteristics of tumor microenvironment. Besides, the accurate tumor diagnosis and visualization of the treatment process are also vital for bacterial therapy. In this paper, a light control engineered bacteria system based on upconversion nanoparticles (UCNP)-mediated time-resolved imaging (TRI) was constructed for colorectal cancer theranostic and therapy. UCNP with different luminous lifetimes were separately modified with the tumor targeting molecule (folic acid) or anaerobic bacteria (Nissle 1917, EcN) to realize the co-localization of tumor tissues, thus improving the diagnostic accuracy based on TRI. In addition, blue light was used to induce engineered bacteria (EcN-pDawn-φx174E/TRAIL) lysis and the release of tumor apoptosis-related inducing ligand (TRAIL), thus triggering tumor cell death. In vitro and in vivo results indicated that this system could achieve accurate tumor diagnosis and light-controlled cancer therapy. EcN-pDawn-φx174E/TRAIL with blue light irradiation could inhibit 53% of tumor growth in comparison to that without blue light irradiation (11.8%). We expect that this engineered bacteria system provides a new technology for intelligent bacterial therapy and the construction of cancer theranostics.

Abstract: Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several 10-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

Abstract: Single-cell behaviors are essential during early-stage biofilm formation. In this study, we aimed to evaluate whether single-cell behaviors could be precisely and continuously manipulated by optogenetics. We thus established adaptive tracking illumination (ATI), a novel illumination method to precisely manipulate the gene expression and bacterial behavior of Pseudomonas aeruginosa on the surface at the single-cell level by using the combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation, and adaptive microscopy. ATI enables precise gene expression control by manipulating the optogenetic module gene expression and type IV pili (TFP)-mediated motility and microcolony formation during biofilm formation through bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) level modifications in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms could be controlled using ATI. Therefore, this novel method we established might markedly answer various questions or resolve problems in microbiology. KEY POINTS: • High-resolution spatial and continuous optogenetic control of individual bacteria. • Phenotype-specific optogenetic control of individual bacteria. • Capacity to control biologically relevant processes in engineered single cells.