Curated Optogenetic Publication Database

Abstract: Bovine parainfluenza virus type 3 (BPIV3) is a promising vaccine vector against various respiratory virus infections, including the human PIV3, respiratory syncytial virus, and severe acute respiratory syndrome-coronavirus 2 infections. In this study, we combined the Magnet system and reverse genetic approach to generate photocontrollable BPIV3. An optically controllable Magnet gene was inserted into the H2 region of the BPIV3 large protein gene, which encodes an RNA-dependent RNA polymerase. The generated photocontrollable BPIV3 grew in specific regions of the cell sheet only when illuminated with blue light, suggesting that spatiotemporal control can aid in safe clinical applications of BPIV3.

Abstract: To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to “lock” onto a particular direction, limiting the ability of cells to reorient. We use spatially-defined optogenetic control of a leading edge organizer (PI3K) to probe how cells balance “decisiveness” needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibitor that destabilizes the leading edge to promote exploration. We show that this local inhibitor enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals.

Abstract: Cell communication is a widespread mechanism in biology, allowing the transmission of information about environmental conditions. In order to understand how cell communication modulates relevant biological processes such as survival, division, differentiation, and apoptosis, different synthetic systems based on chemical induction have been successfully developed. In this work, we coupled cell communication and optogenetics in the budding yeast Saccharomyces cerevisiae. Our approach is based on two strains connected by the light-dependent production of α-factor pheromone in one cell type, which induces gene expression in the other type. After the individual characterization of the different variants of both strains, the optogenetic intercellular system was evaluated by combining the cells under contrasting illumination conditions. Using luciferase as a reporter gene, specific co-cultures at a 1:1 ratio displayed activation of the response upon constant blue light, which was not observed for the same cell mixtures grown in darkness. Then, the system was assessed at several dark/blue-light transitions, where the response level varies depending on the moment in which illumination was delivered. Furthermore, we observed that the amplitude of response can be tuned by modifying the initial ratio between both strains. Finally, the two-population system showed higher fold inductions in comparison with autonomous strains. Altogether, these results demonstrated that external light information is propagated through a diffusible signaling molecule to modulate gene expression in a synthetic system involving microbial cells, which will pave the road for studies allowing optogenetic control of population-level dynamics.

Abstract: The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.

Abstract: Pluripotent stem cells (PSCs) hold great promise for cell-based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light-inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease-deficient CRISPR-associated protein 9 for induced PSCs reprogramming. This system enables remote, non-invasive optogenetical regulation of endogenous Sox2 and Oct4 loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSCLIRE ) under light-emitting diode-based illumination. iPSCLIRE cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSCLIRE cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non-invasive control of user-defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non-invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications.

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: Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can act as repressors or activators, how these functions are implemented at the molecular level has remained elusive. Here we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 minute) and memoryless. Finally, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Abstract: Photosensory protein domains are the basis of optogenetic protein engineering. These domains originate from natural sources where they fulfill specific functions ranging from the protection against photooxidative damage to circadian rhythms. When used in synthetic biology, the features of these photosensory domains can be specifically tailored towards the application of interest, enabling their full exploitation for optogenetic regulation in basic research and applied bioengineering. In this work, we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity. We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants. For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted. We functionally characterize the variants in vivo in bacteria on the single-cell and the population levels. We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells. We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments. Our findings expand the applicability of the widely used Magnets photosensors by enabling a tuning towards the needs of specific optogenetic regulation strategies. More generally, our approach will aid optogenetic approaches by making the adaptation of photosensor properties possible to better suit specific experimental or bioprocess needs.

Abstract: In the budding yeast Saccharomyces cerevisiae, the FUN-LOV (FUNgal Light Oxygen and Voltage) optogenetic switch enables high levels of light-activated gene expression in a reversible and tunable fashion. The FUN-LOV components, under identical promoter and terminator sequences, are encoded in two different plasmids, which limits its future applications in wild and industrial yeast strains. In this work, we aim to expand the molecular versatility of the FUN-LOV switch to increase its biotechnological applications. Initially, we generated new variants of this system by replacing the promoter and terminator sequences and by cloning the system in a single plasmid (FUN-LOVSP). In a second step, we included the nourseothricin (Nat) or hygromycin (Hph) antibiotic resistances genes in the new FUN-LOVSP plasmid, generating two new variants (FUN-LOVSP-Nat and FUN-LOVSP-Hph), to allow selection after genome integration. Then, we compared the levels of light-activated expression for each FUN-LOV variants using the luciferase reporter gene in the BY4741 yeast strain. The results indicate that FUN-LOVSP-Nat and FUN-LOVSP-Hph, either episomally or genome integrated, reached higher levels of luciferase expression upon blue-light stimulation compared the original FUN-LOV system. Finally, we demonstrated the functionality of FUN-LOVSP-Hph in the 59A-EC1118 wine yeast strain, showing similar levels of reporter gene induction under blue-light respect to the laboratory strain, and with lower luciferase expression background in darkness condition. Altogether, the new FUN-LOV variants described here are functional in different yeast strains, expanding the biotechnological applications of this optogenetic tool.

Abstract: The production of recombinant proteins is a problem of major industrial and pharmaceutical importance. Secretion of the protein by the host cell considerably simplifies downstream purification processes. However, it is also the limiting production step for many hard‐to‐secrete proteins. Current solutions involve extensive chassis engineering to favor trafficking and limit protein degradation triggered by excessive secretion‐ associated stress. Here, we propose instead a regulation‐based strategy in which induction is dynamically adjusted based on the current stress level of the cells. Using a small collection of hard‐to‐secrete proteins and a bioreactor‐based platform with automated cytometry measurements, we demonstrate that the regulation sweet spot is indicated by the appearance of a bimodal distribution of internal protein and of secretory stress levels, when a fraction of the cell population accumulates high amounts of proteins, decreases growth, and faces significant stress, that is, experiences a secretion burn‐out. In these cells, adaptations capabilities are overwhelmed by a too strong production. With these notions, we define an optimal stress level based on physiological readouts. Then, using real‐time control, we demonstrate that a strategy that keeps the stress at optimal levels increases production of a single‐chain antibody by 70%.

Abstract: Gene expression is inherently dynamic, due to complex regulation and stochastic biochemical events. However, the effects of these dynamics on cell phenotypes can be difficult to determine. Researchers have historically been limited to passive observations of natural dynamics, which can preclude studies of elusive and noisy cellular events where large amounts of data are required to reveal statistically significant effects. Here, using recent advances in the fields of machine learning and control theory, we train a deep neural network to accurately predict the response of an optogenetic system in Escherichia coli cells. We then use the network in a deep model predictive control framework to impose arbitrary and cell-specific gene expression dynamics on thousands of single cells in real time, applying the framework to generate complex time-varying patterns. We also showcase the framework’s ability to link expression patterns to dynamic functional outcomes by controlling expression of the tetA antibiotic resistance gene. This study highlights how deep learning-enabled feedback control can be used to tailor distributions of gene expression dynamics with high accuracy and throughput.

Abstract: Certain anaerobic microbes with the capability to colonize in tumor microenvironment tend to express the heterologous gene in a sustainable manner, which would inevitably comprise the therapeutic efficacy and induce off-tumor toxicity in vivo. To improve the therapeutic precision and controllability of bacteria-based therapeutics, Escherichia coli Nissle 1917 (EcN) engineered to sense blue light and release the encoded flagellin B (flaB), is conjugated with lanthanide upconversion nanoparticles (UCNPs) for near-infrared (NIR) nano-optogenetic cancer immunotherapy. Upon 808 nm photoirradiation, UCNPs emit at the blue region to photoactivate the EcN for secretion of flaB, which subsequently binds to Toll-like receptor 5 expressed on the membrane of macrophages for activating immune response via MyD88-dependent signal pathway. Such synergism leads to significant tumor regression in different tumor models and metastatic tumors with negligible side effects. Our studies based on NIR nano-optogenetic platform highlight the rational of leveraging the optogenetic tools combined natural propensity of certain bacteria for cancer immunotherapy. This article is protected by copyright. All rights reserved.

Abstract: The experimental need to engineer the genome both in time and space, has led to the development of several photoactivatable Cre recombinase systems. However, the combination of inefficient and non-intentional background recombination has prevented thus far the wide application of these systems in biological and biomedical research. Here, we engineer an optimized photoactivatable Cre recombinase system that we refer to as doxycycline- and light-inducible Cre recombinase (DiLiCre). Following extensive characterization in cancer cell and organoid systems, we generate a DiLiCre mouse line, and illustrated the biological applicability of DiLiCre for light-induced mutagenesis in vivo and positional cell-tracing by intravital microscopy. These experiments illustrate how newly formed HrasV12 mutant cells follow an unnatural movement towards the interfollicular dermis. Together, we develop an efficient photoactivatable Cre recombinase mouse model and illustrate how this model is a powerful genome-editing tool for biological and biomedical research.

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

Abstract: The spatial organization of cell-surface receptors is fundamental for the coordination of biological responses to physical and biochemical cues of the extracellular matrix. How serine/threonine kinase receptors, ALK3-BMPRII, cooperate with integrins upon BMP2 to drive cell migration is unknown. Whether the dynamics between integrins and BMP receptors intertwine in space and time to guide adhesive processes is yet to be elucidated. We found that BMP2 stimulation controls the spatial organization of BMPRs by segregating ALK3 from BMPRII into β3 integrin-containing focal adhesions. The selective recruitment of ALK3 to focal adhesions requires β3 integrin engagement and ALK3 activation. BMP2 controls the partitioning of immobilized ALK3 within and outside focal adhesions according to single-protein tracking and super-resolution imaging. The spatial control of ALK3 in focal adhesions by optogenetics indicates that ALK3 acts as an adhesive receptor by eliciting cell spreading required for cell migration. ALK3 segregation from BMPRII in integrin-based adhesions is a key aspect of the spatio-temporal control of BMPR signaling.

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: A wide array of optogenetic tools is available that allow for precise spatiotemporal control over many cellular processes. These tools have been especially popular among zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and apoptosis.

Abstract: The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Abstract: The ability to drive expression of exogenous genes in different tissues and cell types, under the control of specific enhancers, has been crucial for discovery in biology. While many enhancers drive expression broadly, several genetic tools were developed to obtain access to isolated cell types. Studies of spatially organized neuropiles in the central nervous system of fruit flies have raised the need for a system that targets subsets of cells within a single neuronal type, a feat currently dependent on stochastic flip-out methods. To access the same cells within a given expression pattern consistently across fruit flies, we developed the light-gated expression system LOV-LexA. We combined the bacterial LexA transcription factor with the plant-derived light, oxygen, or voltage photosensitive domain and a fluorescent protein. Exposure to blue light uncages a nuclear localizing signal in the C-terminal of the light, oxygen, or voltage domain and leads to the translocation of LOV-LexA to the nucleus, with the subsequent initiation of transcription. LOV-LexA enables spatial and temporal control of expression of transgenes under LexAop sequences in larval fat body and pupal and adult neurons with blue light. The LOV-LexA tool is ready to use with GAL4 and Split-GAL4 drivers in its current form and constitutes another layer of intersectional genetics that provides light-controlled genetic access to specific cells across flies.

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.

Abstract: 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.

Abstract: BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR (inverse-BAR) domain containing tools derived from the fusion of the A. thaliana Cryptochrome 2 photoreceptor and I-BAR protein domains ('CRY-BARs') with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate here that the CRY-BAR optogenetic tool evokes membrane dynamics changes associated with cellular activity. Moreover, we provide evidence that ezrin, an actin and PIP2 binding protein, acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, we propose that CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.

Abstract: Communities of microbes play important roles in natural environments and hold great potential for deploying division-of-labor strategies in synthetic biology and bioproduction. However, the difficulty of controlling the composition of microbial consortia over time hinders their optimal use in many applications. Here, we present a fully automated, high-throughput platform that combines real-time measurements and computer-controlled optogenetic modulation of bacterial growth to implement precise and robust compositional control of a two-strain E. coli community. In addition, we develop a general framework for dynamic modeling of synthetic genetic circuits in the physiological context of E. coli and use a host-aware model to determine the optimal control parameters of our closed-loop compositional control system. Our platform succeeds in stabilizing the strain ratio of multiple parallel co-cultures at arbitrary levels and in changing these targets over time, opening the door for the implementation of dynamic compositional programs in synthetic bacterial communities.

Abstract: Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to control gene expression independently from mammalian transcriptional and/or translational machineries thus working orthogonally to endogenous regulatory mechanisms. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize, and optimize a new optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool is orthogonal to the cellular machinery for transcription and consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7-mOptoT7). In our study we exploited the T7 polymerase's viral origins to tune our system's expression level, reaching up to an almost 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition, and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate the gene expression burden when compared to another optogenetic construct. These properties make mOptoT7 a powerful new tool to use when orthogonality and viral RNA species (that lack endogenous RNA modifications) are desired.