Showing 1 - 25 of 381 results
Selective induction of programmed cell death using synthetic biology tools.
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.
Design principles for engineering light-controlled antibodies.
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.
Remotely Controllable Engineered Bacteria for Targeted Therapy of Pseudomonas aeruginosa Infection.
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.
A biological camera that captures and stores images directly into DNA.
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.
Multidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor.
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.
A photoreceptor-based hydrogel with red light-responsive reversible sol-gel transition as transient cellular matrix.
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 yielded hydrogels, where biological photoreceptors translated optical signals into a reversible and adjustable change in hydrogel mechanics. While their initial application provided important insights into mechanobiology, broader implementation is limited by a small dynamic range of addressable stiffness. Here, we overcome this limitation 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. We comprehensively characterize the hydrogel’s light-responsive mechanical properties and apply it as reversible extracellular matrix for the spatiotemporally controlled deposition of mammalian cells within a microfluidic chip. We anticipate that this technology will open new avenues for the site- and time-specific positioning of cells and will contribute to overcome spatial restrictions.
Optogenetic Methods in Plant Biology.
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.
The clinical potential of optogenetic interrogation of pathogenesis.
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.
Genetically encoded imaging tools for investigating cell dynamics at a glance.
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.
PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae.
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.
Live Imaging with Genetically Encoded Physiologic Sensors and Optogenetic Tools.
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.
Opto-APC: Engineering of cells that display phytochrome B on their surface for optogenetic studies of cell-cell interactions.
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.
Optical Control of Cell Signaling with Red/Far-Red Light-Responsive Optogenetic Tools in Caenorhabditis elegans.
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.
Programming the lifestyles of engineered bacteria for cancer therapy.
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.
Measurement of Secreted Embryonic Alkaline Phosphatase.
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.
A red light-controlled probiotic bio-system for in-situ gut-brain axis regulation.
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.
Orthogonal Light-Dependent Membrane Adhesion Induces Social Self-Sorting and Member-Specific DNA Communication in Synthetic Cell Communities.
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.
Using optogenetics to investigate the shared mechanisms of apical-basal polarity and mitosis.
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.
Retraction: "Long noncoding RNA ZFPM2-AS1 is involved in lung adenocarcinoma via miR-511-3p/AFF4 pathway," by Juan Li, Jun Ge, Ye Yang, Bin Liu, Min Zheng, and Rui Shi, J Cell Biochem. 2020; 2534-2542: The above article, published online on November 6, 2019, in Wiley Online Library (https://doi.org/10.1002/jcb.29476) has been retracted by agreement between the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The retraction has been agreed after the authors stated that unintentional errors occurred during the research process, and the experimental results cannot be verified. Thus, the conclusions are considered to be invalid. The authors were not available for a final confirmation of the retraction.
Optogenetic-controlled immunotherapeutic designer cells for post-surgical cancer immunotherapy.
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.
Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives.
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.
An adaptive tracking illumination system for optogenetic control of single bacterial cells.
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.
Ligand-independent receptor clustering modulates transmembrane signaling: a new paradigm.
Cell-surface receptors mediate communication between cells and their environment. Lateral membrane organization and dynamic receptor cluster formation are fundamental in signal transduction and cell signaling. However, it is not yet fully understood how receptor clustering modulates a wide variety of physiologically relevant processes. Recent growing evidence indicates that biological responses triggered by membrane receptors can be modulated even in the absence of the natural receptor ligand. We review the most recent findings on how ligand-independent receptor clustering can regulate transmembrane signaling. We discuss the latest technologies to control receptor assembly, such as DNA nanotechnology, optogenetics, and optochemistry, focusing on the biological relevance and unraveling of ligand-independent signaling.
The bright frontiers of microbial metabolic optogenetics.
In recent years, light-responsive systems from the field of optogenetics have been applied to several areas of metabolic engineering with remarkable success. By taking advantage of light's high tunability, reversibility, and orthogonality to host endogenous processes, optogenetic systems have enabled unprecedented dynamical controls of microbial fermentations for chemical production, metabolic flux analysis, and population compositions in co-cultures. In this article, we share our opinions on the current state of this new field of metabolic optogenetics.We make the case that it will continue to impact metabolic engineering in increasingly new directions, with the potential to challenge existing paradigms for metabolic pathway and strain optimization as well as bioreactor operation.
Optogenetic Control of Bacterial Expression by Red Light.
In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and far-red light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.