Curated Optogenetic Publication Database

Abstract: Light has become established as a tool not only to visualize and investigate but also to steer biological systems. This review starts by discussing the unique features that make light such an effective control input in biology. It then gives an overview of how light‐control came to progress, starting with photoactivatable compounds and leading up to current genetic implementations using optogenetic approaches. The review then zooms in on optogenetics, focusing on photosensitive proteins, which form the basis for optogenetic engineering using synthetic biological approaches. As the regulation of transcription provides a highly versatile means for steering diverse biological functions, the focus of this review then shifts to transcriptional light regulators, which are presented in the biotechnologically highly relevant model organism Escherichia coli.

Abstract: Living organisms have evolved sophisticated cell-mediated biomineralization mechanisms to build structurally ordered, environmentally adaptive composite materials. Despite advances in biomimetic mineralization research, it remains difficult to produce mineralized composites that integrate the structural features and 'living' attributes of their natural counterparts. Here, inspired by natural graded materials, we developed living patterned and gradient composites by coupling light-inducible bacterial biofilm formation with biomimetic hydroxyapatite (HA) mineralization. We showed that both the location and the degree of mineralization could be regulated by tailoring functional biofilm growth with spatial and biomass density control. The cells in the composites remained viable and could sense and respond to environmental signals. Additionally, the composites exhibited a maximum 15-fold increase in Young's modulus after mineralization and could be applied to repair damage in a spatially controlled manner. Beyond insights into the mechanism of formation of natural graded composites, our study provides a viable means of fabricating living composites with dynamic responsiveness and environmental adaptability.

Abstract: Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.

Abstract: Optogenetics harnesses natural photoreceptors to non-invasively control selected processes in cells with previously unmet spatiotemporal precision. Linking the activity of a protein of choice to the conformational state of a photosensor domain through allosteric coupling represents a powerful method for engineering light-responsive proteins. It enables the design of compact and highly potent single-component optogenetic systems with fast on- and off-switching kinetics. However, designing protein-photoreceptor chimeras, in which structural changes of the photoreceptor are effectively propagated to the fused effector protein, is a challenging engineering problem and often relies on trial and error. Here, recent advances in the design and application of optogenetic allosteric switches are reviewed. First, an overview of existing optogenetic tools based on inducible allostery is provided and their utility for cell biology applications is highlighted. Focusing on light-oxygen-voltage domains, a widely applied class of small blue light sensors, the available strategies for engineering light-dependent allostery are presented and their individual advantages and limitations are highlighted. Finally, high-throughput screening technologies based on comprehensive insertion libraries, which could accelerate the creation of stimulus-responsive receptor-protein chimeras for use in optogenetics and beyond, are discussed.

Abstract: Chemical molecules specifically secreted into the blood and targeted tissues by intestinal microbiota can effectively affect the associated functions of the intestine especially immunity, representing a new strategy for immune-related diseases. However, proper ways of regulating the secretion metabolism of specific strains still remain to be established. In this article, an upconversion optogenetic micro-nanosystem was constructed to effectively regulate the specific secretion of engineered bacteria. The system included two major modules: (i) Modification of secretory light-responsive engineered bacteria. (ii) Optical sensing mediated by upconversion optogenetic micro-nanosystem. This system could regulate the efficient secretion of immune factors by engineered bacteria through optical manipulation. Inflammatory bowel disease and subcutaneously transplanted tumors were selected to verify the effectiveness of the system. Our results showed that the endogenous factor TGF-β1 could be controllably secreted to suppress the intestinal inflammatory response. Additionally, regulatory secretion of IFN-γ was promoted to slow the progression of B16F10 tumor.

Abstract: Control of the lac operon with isopropyl β-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.

Abstract: Optogenetics and photonic technologies are changing the future of medicine. To implement light‐based therapies in the clinic, patient‐friendly devices that can deliver light inside the body while offering tunable properties and compatibility with soft tissues are needed. Here extrusion printing of degradable, hydrogel‐based optical waveguides with optical losses as low as 0.1 dB cm−1 at visible wavelengths is described. Core‐only and core‐cladding fibers are printed at room temperature from polyethylene glycol (PEG)‐based and PEG/Pluronic precursors, and cured by in situ photopolymerization. The obtained waveguides are flexible, with mechanical properties tunable within a tissue‐compatible range. Degradation times are also tunable by adjusting the molar mass of the diacrylate gel precursors, which are synthesized by linking PEG diacrylate (PEGDA) with varying proportions of DL‐dithiothreitol (DTT). The printed waveguides are used to activate photochemical and optogenetic processes in close‐to‐physiological environments. Light‐triggered migration of cells in a photoresponsive 3D hydrogel and drug release from an optogenetically‐engineered living material by delivering light across >5 cm of muscle tissue are demonstrated. These results quantify the in vitro performance, and reflect the potential of the printed degradable fibers for in vivo and clinical applications.

Abstract: The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

Abstract: Phototropins are photoreceptor proteins, which regulate blue light dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (Light-Oxygen-Voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism which leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from Chlamydomonas reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that the adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. Particularly, the change in conformation and associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the β-sheet, ultimately driving a change in electrostatic potential on the protein surface. Based on the change of electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from Chlamydomonas reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.

Abstract: Cell-free systems, as part of the synthetic biology field, have become a critical platform in biological studies. However, there is a lack of research into developing a switch for a dynamical control of the transcriptional and translational process. The optogenetic tool has been widely proven as an ideal control switch for protein synthesis due to its nontoxicity and excellent time-space conversion. Hence, in this study, a blue light-regulated two-component system named YF1/FixJ was incorporated into an Escherichia coli-based cell-free system to control protein synthesis. The corresponding cell-free system successfully achieved a 5-fold dynamic protein expression by blue light repression and 3-fold dynamic expression by blue light activation. With the aim of expanding the applications of cell-free synthetic biology, the cell-free blue light-sensing system was used to perform imaging, light-controlled antibody synthesis, and light-triggered artificial cell assembly. This study can provide a guide for further research into the field of cell-free optical sensing. Moreover, it will also promote the development of cell-free synthetic biology and optogenetics through applying the cell-free optical sensing system to synthetic biology education, biopharmaceutical research, and artificial cell construction.

Abstract: Light-regulated modules offer unprecedented new ways to control cellular behaviour with precise spatial and temporal resolution. Among a variety of bacterial light-switchable gene expression systems, single-component systems consisting of single transcription factors would be more useful due to the advantages of speed, simplicity, and versatility. In the present study, we developed a single-component light-activated bacterial gene expression system (eLightOn) based on a novel LOV domain from Rhodobacter sphaeroides (RsLOV). The eLightOn system showed significant improvements over the existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability. Additionally, the induction characteristics, including regulatory windows, activation kinetics and light sensitivities, were highly tunable by altering the expression level of LexRO. We demonstrated the usefulness of the eLightOn system in regulating cell division and swimming by controlling the expression of the FtsZ and CheZ genes, respectively, as well as constructing synthetic Boolean logic gates using light and arabinose as the two inputs. Taken together, our data indicate that the eLightOn system is a robust and highly tunable tool for quantitative and spatiotemporal control of bacterial gene expression.

Abstract: In optogenetics, light-sensitive proteins are specifically expressed in target cells and light is used to precisely control the activity of these proteins at high spatiotemporal resolution. Optogenetics initially used naturally occurring photoreceptors to control neural circuits, but has expanded to include carefully designed and engineered photoreceptors. Several optogenetic constructs are based on plant photoreceptors, but their application to plant systems has been limited. Here, we present perspectives on the development of plant optogenetics, considering different levels of design complexity. We discuss how general principles of light-driven signal transduction can be coupled with approaches for engineering protein folding to develop novel optogenetic tools. Finally, we explore how the use of computation, networks, circular permutation, and directed evolution could enrich optogenetics.

Abstract: Two-component systems (TCS) constitute the predominant means by which prokaryotes read out and adapt to their environment. Canonical TCSs comprise a sensor histidine kinase (SHK), usually a transmembrane receptor, and a response regulator (RR). In signal-dependent manner, the SHK autophosphorylates and in turn transfers the phosphoryl group to the RR which then elicits downstream responses, often in form of altered gene expression. SHKs also catalyze the hydrolysis of the phospho-RR, hence, tightly adjusting the overall degree of RR phosphorylation. Photoreceptor histidine kinases are a subset of mostly soluble, cytosolic SHKs that sense light in the near-ultraviolet to near-infrared spectral range. Owing to their experimental tractability, photoreceptor histidine kinases serve as paradigms and provide unusually detailed molecular insight into signal detection, decoding, and regulation of SHK activity. The synthesis of recent results on receptors with light-oxygen-voltage, bacteriophytochrome and microbial rhodopsin sensor units identifies recurring, joint signaling strategies. Light signals are initially absorbed by the sensor module and converted into subtle rearrangements of α helices, mostly through pivoting and rotation. These conformational transitions propagate through parallel coiled-coil linkers to the effector unit as changes in left-handed superhelical winding. Within the effector, subtle conformations are triggered that modulate the solvent accessibility of residues engaged in the kinase and phosphatase activities. Taken together, a consistent view of the entire trajectory from signal detection to regulation of output emerges. The underlying allosteric mechanisms could widely apply to TCS signaling in general.

Abstract: The fast-growing non-model marine bacterium Vibrio natriegens has recently garnered attention as a host for molecular biology and biotechnology applications. In order further its capabilities as a synthetic biology chassis, we have characterized a wide range of genetic parts and tools for use in V. natriegens. These parts include many commonly-used resistance markers, promoters, ribosomal binding sites, reporters, terminators, degradation tags, origin of replication sequences and plasmid backbones. We have characterized the behavior of these parts in different combinations and have compared their functionality in V. natriegens and Escherichia coli. Plasmid stability over time, plasmid copy numbers, and production load on the cells were also evaluated. Additionally, we tested constructs for chemical and optogenetic induction and characterized basic engineered circuit behavior in V. natriegens. The results indicate that while most parts and constructs work similarly in the two organisms, some deviate significantly. Overall, these results will serve as a primer for anyone interested in engineering V. natriegens and will aid in developing more robust synthetic biology principles and approaches for this non-model chassis.

Abstract: Living cells can impart materials with advanced functions, such as sense-and-respond, chemical production, toxin remediation, energy generation and storage, self-destruction, and self-healing. Here, an approach is presented to use light to pattern Escherichia coli onto diverse materials by controlling the expression of curli fibers that anchor the formation of a biofilm. Different colors of light are used to express variants of the structural protein CsgA fused to different peptide tags. By projecting color images onto the material containing bacteria, this system can be used to pattern the growth of composite materials, including layers of protein and gold nanoparticles. This is used to pattern cells onto materials used for 3D printing, plastics (polystyrene), and textiles (cotton). Further, the adhered cells are demonstrated to respond to sensory information, including small molecules (IPTG and DAPG) and light from light-emitting diodes. This work advances the capacity to engineer responsive living materials in which cells provide diverse functionality.

Abstract: Living organisms exhibit a wide range of intrinsic adaptive responses to incident light. Likewise, in optogenetics, biological systems are tailored to initiate predetermined cellular processes upon light exposure. As genetically encoded, light-gated actuators, sensory photoreceptors are at the heart of these responses in both the natural and engineered scenarios. Upon light absorption, photoreceptors enter a series of generally rapid photochemical reactions leading to population of the light-adapted signaling state of the receptor. Notably, this state persists for a while before thermally reverting to the original dark-adapted resting state. As a corollary, the inactivation of photosensitive biological circuits upon light withdrawal can exhibit substantial inertia. Intermittent illumination of suitable pulse frequency can hence maintain the photoreceptor in its light-adapted state while greatly reducing overall light dose, thereby mitigating adverse side effects. Moreover, several photoreceptor systems may be actuated sequentially with a single light color if they sufficiently differ in their inactivation kinetics. Here, we detail the construction of programmable illumination devices for the rapid and parallelized testing of biological responses to diverse lighting regimes. As the technology is based on open electronics and readily available, inexpensive components, it can be adopted by most laboratories at moderate expenditure. As we exemplify for two use cases, the programmable devices enable the facile interrogation of diverse illumination paradigms and their application in optogenetics and photobiology.

Abstract: On-demand and long-term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self-replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin-free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light-regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.

Abstract: Developing materials to encapsulate and deliver functional proteins inside the body is a challenging yet rewarding task for therapeutic purposes. High production costs, mostly associated with the purification process, short-term stability in vivo, and controlled and prolonged release are major hurdles for the clinical application of protein-based biopharmaceuticals. In an attempt to overcome these hurdles, herein, the possibility of incorporating bacteria as protein factories into a material and externally controlling protein release using optogenetics is demonstrated. By engineering bacteria to express and secrete a red fluorescent protein in response to low doses of blue light irradiation and embedding them in agarose hydrogels, living materials are fabricated capable of releasing proteins into the surrounding medium when exposed to light. These bacterial hydrogels allow spatially confined protein expression and dosed protein release over several weeks, regulated by the area and extent of light exposure. The possibility of incorporating such complex functions in a material using relatively simple material and genetic engineering strategies highlights the immense potential and versatility offered by living materials for protein-based biopharmaceutical delivery.

Abstract: Synthetic biology has emerged as a multidisciplinary field that provides new tools and approaches to address longstanding problems in biology. It integrates knowledge from biology, engineering, mathematics, and biophysics to build—rather than to simply observe and perturb—biological systems that emulate natural counterparts or display novel properties. The interface between synthetic and developmental biology has greatly benefitted both fields and allowed to address questions that would remain challenging with classical approaches due to the intrinsic complexity and essentiality of developmental processes. This Progress Report provides an overview of how synthetic biology can help to understand a process that is crucial for the development of multicellular organisms: pattern formation. It reviews the major mechanisms of genetically encoded synthetic systems that have been engineered to establish spatial patterns at the population level. Limitations, challenges, applications, and potential opportunities of synthetic pattern formation are also discussed.

Abstract: Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture.

Abstract: Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct-pDawn-Ag43-that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 μm can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

Abstract: Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Abstract: Optogenetics is a powerful method for studying dynamic processes in living cells and has advanced cell biology research over the recent past. Key to the successful application of optogenetics is the careful design of the light‐sensing module, typically employing a natural or engineered photoreceptor that links the exogenous light input to the cellular process under investigation. Light–oxygen–voltage (LOV) domains, a highly diverse class of small blue light sensors, have proven to be particularly versatile for engineering optogenetic input modules. These can function via diverse modalities, including inducible allostery, protein recruitment, dimerization, or dissociation. This study reviews recent advances in the development of LOV domain‐based optogenetic tools and their application for studying and controlling selected cellular functions. Focusing on the widely employed LOV2 domain from Avena sativa phototropin‐1, this review highlights the broad spectrum of engineering opportunities that can be explored to achieve customized optogenetic regulation. Finally, major bottlenecks in the development of optogenetic methods are discussed and strategies to overcome these with recent synthetic biology approaches are pointed out.

Abstract: In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occuring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest. Immediate advantages of these new photoresponsive proteins such as genetic encoding, no requirement of chemical modification, and reversibility are paid by difficulties in predicting the envisaged activity or type and site of domain fusion. In this article, we summarize recent advances and give a survey on currently available design concepts for engineering photoswitchable proteins.

Abstract: Intracellular products (e.g., insulin), which are obtained through cell lysis, take up a big share of the biotech industry. It is often time-consuming, laborious, and environment-unfriendly to disrupt bacterial cells with traditional methods. In this study, we developed a molecular device for controlling cell lysis with light. We showed that intracellular expression of a single lysin protein was sufficient for efficient bacterial cell lysis. By placing the lysin-encoding gene under the control of an improved light-controlled system, we successfully controlled cell lysis by switching on/off light: OD600 of the Escherichia coli cell culture was decreased by twofold when the light-controlled system was activated under dark condition. We anticipate that our work would not only pave the way for cell lysis through a convenient biological way in fermentation industry, but also provide a paradigm for applying the light-controlled system in other fields of biotech industry.