Curated Optogenetic Publication Database

Abstract: Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.

Abstract: The extracellular matrix (ECM) comprises a meshwork of biomacromolecules whose composition, architecture, and macroscopic properties, such as mechanics, instruct cell fate decisions during development and disease progression. Current methods implemented in mechanotransduction studies either fail to capture real-time mechanical dynamics or utilize synthetic polymers that lack the fibrillar nature of their natural counterparts. Here we present an optogenetic-inspired tool to construct light-responsive ECM mimetic hydrogels comprised exclusively of natural ECM proteins. Optogenetic tools offer seconds temporal resolution and submicron spatial resolution, permitting researchers to probe cell signaling dynamics with unprecedented precision. Here we demonstrated our approach of using SNAP-tag and its thiol-targeted substrate, benzylguanine-maleimide, to covalently attach blue-light-responsive proteins to collagen hydrogels. The resulting material (OptoGel), in addition to encompassing the native biological activity of collagen, stiffens upon exposure to blue light and softens in the dark. Optogels have immediate use in dissecting the cellular response to acute mechanical inputs and may also have applications in next-generation biointerfacing prosthetics.

Abstract: Microbial synthesis of chemicals typically requires the redistribution of metabolic flux toward the synthesis of targeted products. Dynamic control is emerging as an effective approach for solving the hurdles mentioned above. As light could control the cell behavior in a spatial and temporal manner, the optogenetic-CRISPR interference (opto-CRISPRi) technique that allocates the metabolic resources according to different optical signal frequencies will enable bacteria to be controlled between the growth phase and the production stage. In this study, we applied a blue light-sensitive protein EL222 to regulate the expression of the dCpf1-mediated CRISPRi system that turns off the competitive pathways and redirects the metabolic flux toward the heterologous muconic acid synthesis in Escherichia coli. We found that the opto-CRISPRi system dynamically regulating the suppression of the central metabolism and competitive pathways could increase the muconic acid production by 130%. These results demonstrated that the opto-CRISPRi platform is an effective method for enhancing chemical synthesis with broad utilities.

Abstract: The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in Saccharomyces cerevisiae. The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible GAL1 promoter (PGAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with PGAL1-S achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and PGAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.

Abstract: Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.

Abstract: Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.

Abstract: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGFβ) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic, and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise activation of the BMP signaling pathway. Here, we report the development of an optogenetic BMP signaling system (optoBMP) that enables rapid induction of the canonical BMP signaling pathway driven by illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signaling pathway through variable light stimulation. Optogenetic control of BMP signaling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.

Abstract: Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Abstract: The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A. thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.

Abstract: Multi-objective optimization of microbial chassis for the production of xenobiotic compounds requires the implementation of metabolic control strategies that permit dynamic distribution of cellular resources between biomass and product formation. We addressed this need in a previous study by engineering the T7 RNA polymerase to be thermally responsive. The modified polymerase is activated only after the temperature of the host cell falls below 18oC, and Escherichia coli cells that employ the protein to transcribe the heterologous lycopene biosynthetic pathway exhibit impressive improvements in productivity. We have expanded our toolbox of metabolic switches in the current study by engineering a version of the T7 RNA polymerase that drives the transition between biomass and product formation upon stimulation with red light. The engineered polymerase is expressed as two distinct polypeptide chains. Each chain comprises one of two photoactive components from Arabidopsis thaliana, phytochrome B (PhyB) and phytochrome-integrating factor 3 (PIF3), as well as the N- or C-terminus domains of both, the vacuolar ATPase subunit (VMA) intein of Saccharomyces cerevisiae and the polymerase. Red light drives photodimerization of PhyB and PIF3, which then brings together the N- and C-terminus domains of the VMA intein. Trans-splicing of the intein follows suit and produces an active form of the polymerase that subsequently transcribes any sequence that is under the control of a T7 promoter. The photodimerization also involves a third element, the cyanobacterial chromophore phycocyanobilin (PCB), which too is expressed heterologously by E. coli. We deployed this version of the T7 RNA polymerase to control the production of lycopene in E. coli and observed tight control of pathway expression. We tested a variety of expression configurations to identify one that imposes the lowest metabolic burden on the strain, and we subsequently optimized key parameters such as the source, moment and duration of photostimulation. We also identified targets for future refinement of the circuit. In summary, our work is a significant advance for the field and greatly expands on previous work by other groups that have used optogenetic circuits to control heterologous metabolism in prokaryotic hosts.

Abstract: The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell-cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell-cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.

Abstract: Methods that allow labeling and tracking of proteins have been instrumental for understanding their function. Traditional methods for labeling proteins include fusion to fluorescent proteins or self-labeling chemical tagging systems such as SNAP-tag or Halo-tag. These latter approaches allow bright fluorophores or other chemical moieties to be attached to a protein of interest through a small fusion tag. In this work, we sought to improve the versatility of self-labeling chemical-tagging systems by regulating their activity with light. We used light-inducible dimerizers to reconstitute a split SNAP-tag (modified human O6-alkylguanine-DNA-alkyltransferase, hAGT) protein, allowing tight light-dependent control of chemical labeling. In addition, we generated a small split SNAP-tag fragment that can efficiently self-assemble with its complement fragment, allowing high labeling efficacy with a small tag. We envision these tools will extend the versatility and utility of the SNAP-tag chemical system for protein labeling applications.

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: We present a new concept for whole-cell biosensors that couples the response to Hg2+ with bioluminescence and bacterial aggregation. This allows us to use the bacterial aggregation to preconcentrate the bioluminescent bacteria at the substrate surface and increase the sensitivity of Hg2+ detection. This whole-cell biosensor combines a Hg2+-sensitive bioluminescence reporter and light-responsive bacterial cell-cell adhesions. We demonstrate that the blue luminescence in response to Hg2+ is able to photoactivate bacterial aggregation, which provides a second readout for Hg2+ detection. In return, the Hg2+-triggered bacterial aggregation leads to faster sedimentation and more efficient formation of biofilms. At low Hg2+ concentrations, the enrichment of the bacteria in biofilms leads to an up to 10-fold increase in the signal. The activation of photoswitchable proteins with biological light is a new concept in optogenetics, and the presented bacterial biosensor design is transferable to other bioluminescent reporters with particular interest for environmental monitoring.

Abstract: Although the fundamental importance and biotechnological potential of multibacterial communities, also called biofilms, are well-known, our ability to control them is limited. We present a new way of dynamically controlling bacteria-bacteria adhesions by using blue light and how these photoswitchable adhesions can be used to regulate multicellularity and associated bacterial behavior. To achieve this, the photoswitchable proteins nMagHigh and pMagHigh were expressed on bacterial surfaces as adhesins to allow multicellular clusters to assemble under blue light and reversibly disassemble in the dark. Regulation of the bacterial cell-cell adhesions with visible light provides unique advantages including high spatiotemporal control, tunability, and noninvasive remote regulation. Moreover, these photoswitchable adhesions make it possible to regulate collective bacterial functions including aggregation, quorum sensing, biofilm formation, and metabolic cross-feeding between auxotrophic bacteria with light. Overall, the photoregulation of bacteria-bacteria adhesions provides a new way of studying bacterial cell biology and will enable the design of biofilms for biotechnological applications.

Abstract: Nanoscale membrane curvature is now understood to play an active role in essential cellular processes such as endocytosis, exocytosis and actin dynamics. Previous studies have shown that membrane curvature can directly affect protein function and intracellular signaling. However, few methods are able to precisely manipulate membrane curvature in live cells. Here, we report the development of a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. For this purpose, we make use of BAR domain proteins, a family of well-studied membrane-remodeling and membrane-sculpting proteins. Specifically, we engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of positive and negative membrane curvature, respectively. Using opto-FBAR, blue light activation results in the formation of tubular membrane invaginations (positive curvature), controllable down to the subcellular level. Using opto-IBAR, blue light illumination results in the formation of membrane protrusions or filopodia (negative curvature). These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells.

Abstract: Cleavage furrow formation during cytokinesis involves extensive membrane remodeling. In the absence of methods to exert dynamic control over these processes, it has been a challenge to examine the basis of this remodeling. Here we used a subcellular optogenetic approach to induce this at will and found that furrow formation is mediated by actomyosin contractility, retrograde plasma membrane flow, localized decrease in membrane tension and endocytosis. FRAP, 4-D imaging and inhibition or upregulation of endocytosis or exocytosis show that ARF6 and Exo70 dependent localized exocytosis supports a potential model for intercellular bridge elongation. TIRF and Super Resolution Radial Fluctuation (SRRF) stream microscopy show localized VAMP2-mediated exocytosis and incorporation of membrane lipids from vesicles into the plasma membrane at the front edge of the nascent daughter cell. Thus, spatially separated but coordinated plasma membrane depletion and addition are likely contributors to membrane remodeling during cytokinetic processes.

Abstract: Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light. To overcome these problems, here, we have developed FRET biosensors with red-shifted excitation and emission wavelengths. We chose mKOκ and mKate2 as the favorable donor and acceptor pair by calculating the Förster distance. By optimizing the order of fluorescent proteins and modulatory domains of the FRET biosensors, we developed a FRET biosensor backbone named "Booster". The performance of the protein kinase A (PKA) biosensor based on the Booster backbone (Booster-PKA) was comparable to that of AKAR3EV, a previously developed FRET biosensor comprising CFP and YFP. For the proof of concept, we first showed simultaneous monitoring of activities of two protein kinases with Booster-PKA and ERK FRET biosensors based on CFP and YFP. Second, we showed monitoring of PKA activation by Beggiatoa photoactivated adenylyl cyclase, an optogenetic generator of cyclic AMP. Finally, we presented PKA activity in living tissues of transgenic mice expressing Booster-PKA. Collectively, the results demonstrate the effectiveness and versatility of Booster biosensors as an imaging tool in vitro and in vivo.

Abstract: Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membrane-less organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membrane-less organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase separation using light.

Abstract: Optogenetic tools can provide direct and programmable control of gene expression. Light-inducible recombinases, in particular, offer a powerful method for achieving precise spatiotemporal control of DNA modification. However, to-date this technology has been largely limited to eukaryotic systems. Here, we develop optogenetic recombinases for Escherichia coli that activate in response to blue light. Our approach uses a split recombinase coupled with photodimers, where blue light brings the split protein together to form a functional recombinase. We tested both Cre and Flp recombinases, Vivid and Magnet photodimers, and alternative protein split sites in our analysis. The optimal configuration, Opto-Cre-Vvd, exhibits strong blue light-responsive excision and low ambient light sensitivity. For this system we characterize the effect of light intensity and the temporal dynamics of light-induced recombination. These tools expand the microbial optogenetic toolbox, offering the potential for precise control of DNA excision with light-inducible recombinases in bacteria.

Abstract: Light-inducible optogenetic systems offer precise spatiotemporal control over a myriad of biologic processes. Unfortunately, current systems are inherently limited by their dependence on external light sources for their activation. Further, the utility of laser/LED-based illumination strategies are often constrained by the need for invasive surgical procedures to deliver such devices and local heat production, photobleaching and phototoxicity that compromises cell and tissue viability. To overcome these limitations, we developed a novel BRET-activated optogenetics (BEACON) system that employs biologic light to control optogenetic tools. BEACON is driven by self-illuminating bioluminescent-fluorescent proteins that generate "spectrally tuned" biologic light via bioluminescence resonance energy transfer (BRET). Notably, BEACON robustly activates a variety of commonly used optogenetic systems in a spatially restricted fashion, and at physiologically relevant time scales, to levels that are achieved by conventional laser/LED light sources.

Abstract: The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e. ho1 and pcyA genes for synthesis of phycocyanobilin, the ccaS/ccaR system from Synechocystis and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by [i] reassembling them together in a single broad host range, standardized vector and [ii] subjecting the non-coding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species i.e. optimization of relative expression levels and upturning of subcomplex stoichiometry.

Abstract: Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light-modulated protein stabilization system (GLIMPSe) to control intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins: mitogen-activated protein kinase phosphatase 3 (MKP3), a negative regulator of the extracellu-lar signal-regulated kinase (ERK) pathway, as well as a constitutively active form of MEK (CA MEK), a positive regulator of the same pathway. Kinetics study showed that light-induced protein stabilization could be achieved within 30 minutes of blue light stimulation. GLIMPSe enables target-independent optogenetic control of protein activities and therefore minimizes the systematic variation embedded within different photoactivatable proteins. Overall, GLIMPSe promises to achieve light-mediated post-translational stabilization of a wide array of target proteins in live cells.

Abstract: While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tissues within the body. To help address this issue, we present a novel tamoxifen-gated photoactivatable split-Cre recombinase optogenetic system, called TamPA-Cre, that features high spatiotemporal control to limit CAR T cell activity to the tumor site. We created and optimized a novel genetic AND gate switch by integrating the features of tamoxifen-dependent nuclear localization and blue-light-inducible heterodimerization of Magnet protein domains (nMag, pMag) into split Cre recombinase. By fusing the cytosol-localizing mutant estrogen receptor ligand binding domain (ERT2) to the N-terminal half of split Cre(2-59aa)-nMag, the TamPA-Cre protein ERT2-CreN-nMag is physically separated from its nuclear-localized binding partner, NLS-pMag-CreC(60-343aa). Without tamoxifen to drive nuclear localization of ERT2-CreN-nMag, the typically high background of the photoactivation system was significantly suppressed. Upon blue light stimulation following tamoxifen treatment, the TamPA-Cre system exhibits sensitivity to low intensity, short durations of blue light exposure to induce robust Cre-loxP recombination efficiency. We finally demonstrate that this TamPA-Cre system can be applied to specifically control localized CAR expression and subsequently T cell activation. As such, we posit that CAR T cell activity can be confined to a solid tumor site by applying an external stimulus, with high precision of control in both space and time, such as light.

Abstract: Phytochromes are important photoreceptors of plants, bacteria, and fungi responsive to light in the red and far-red spectrum. For increasing applications in basic research, synthetic biology, and materials sciences, it is required to recombinantly produce and purify phytochromes in high amounts. An ideal host organism for this purpose is E. coli due to its widespread use, fast growth, and ability for high-cell-density fermentation. Here, we describe the development of a generic platform for the production of phytochromes in E. coli that is compatible with high-cell-density fermentation. We exemplify our approach by the production of the photosensory domains of phytochrome B (PhyB) from A. thaliana and of the cyanobacterial phytochrome 1 (Cph1) from Synechocystis PCC 6803 in the multigram scale per 10 L fermentation run.