Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 25 of 123 results

Lustro: High-Throughput Optogenetic Experiments Enabled by Automation and a Yeast Optogenetic Toolkit.

blue CRY2/CIB1 Magnets S. cerevisiae Transgene expression
ACS Synth Biol, 11 Jul 2023 DOI: 10.1021/acssynbio.3c00215 Link to full text
Abstract: Optogenetic systems use genetically encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light; however, these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to the high-throughput characterization of optogenetic systems across a range of biological systems and applications.

Remotely Controllable Engineered Bacteria for Targeted Therapy of Pseudomonas aeruginosa Infection.

red BphS P. aeruginosa Immediate control of second messengers
ACS Synth Biol, 7 Jul 2023 DOI: 10.1021/acssynbio.2c00655 Link to full text
Abstract: 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.

OptoCRISPRi-HD: Engineering a Bacterial Green-Light-Activated CRISPRi System with a High Dynamic Range.

green CcaS/CcaR E. coli Control of cytoskeleton / cell motility / cell shape Transgene expression
ACS Synth Biol, 22 May 2023 DOI: 10.1021/acssynbio.3c00035 Link to full text
Abstract: The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the optoCRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of optoCRISPRi often exhibit no more than a 10-fold dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light-activated CRISPRi system with a high dynamic range (40 fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, nonessential genes, or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space-time and extensive targets, our study would facilitate further research involving complex gene networks, metabolic flux redirection, or bioprinting.

Self-Regulated and Bidirectional Communication in Synthetic Cell Communities.

blue iLID in vitro Extracellular optogenetics
ACS Nano, 8 May 2023 DOI: 10.1021/acsnano.2c09908 Link to full text
Abstract: Cell-to-cell communication is not limited to a sender releasing a signaling molecule and a receiver perceiving it but is often self-regulated and bidirectional. Yet, in communities of synthetic cells, such features that render communication efficient and adaptive are missing. Here, we report the design and implementation of adaptive two-way signaling with lipid-vesicle-based synthetic cells. The first layer of self-regulation derives from coupling the temporal dynamics of the signal, H2O2, production in the sender to adhesions between sender and receiver cells. This way the receiver stays within the signaling range for the duration sender produces the signal and detaches once the signal fades. Specifically, H2O2 acts as both a forward signal and a regulator of the adhesions by activating photoswitchable proteins at the surface for the duration of the chemiluminescence. The second layer of self-regulation arises when the adhesions render the receiver permeable and trigger the release of a backward signal, resulting in bidirectional exchange. These design rules provide a concept for engineering multicellular systems with adaptive communication.

PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae.

red PhyB/PIF3 S. cerevisiae Endogenous gene expression
ACS Synth Biol, 4 Apr 2023 DOI: 10.1021/acssynbio.2c00517 Link to full text
Abstract: 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.

A Single-Component Optogenetic Gal4-UAS System Allows Stringent Control of Gene Expression in Zebrafish and Drosophila.

blue VVD D. melanogaster in vivo HEK293 Schneider 2 zebrafish in vivo Transgene expression
ACS Synth Biol, 9 Mar 2023 DOI: 10.1021/acssynbio.2c00410 Link to full text
Abstract: The light-regulated Gal4-UAS system has offered new ways to control cellular activities with precise spatial and temporal resolution in zebrafish and Drosophila. However, the existing optogenetic Gal4-UAS systems suffer from having multiple protein components and a dependence on extraneous light-sensitive cofactors, which increase the technical complexity and limit the portability of these systems. To overcome these limitations, we herein describe the development of a novel optogenetic Gal4-UAS system (ltLightOn) for both zebrafish and Drosophila based on a single light-switchable transactivator, termed GAVPOLT, which dimerizes and binds to gene promoters to activate transgene expression upon blue light illumination. The ltLightOn system is independent of exogenous cofactors and exhibits a more than 2400-fold ON/OFF gene expression ratio, allowing quantitative, spatial, and temporal control of gene expression. We further demonstrate the usefulness of the ltLightOn system in regulating zebrafish embryonic development by controlling the expression of lefty1 by light. We believe that this single-component optogenetic system will be immensely useful in understanding the gene function and behavioral circuits in zebrafish and Drosophila.

Optical Control of Cell Signaling with Red/Far-Red Light-Responsive Optogenetic Tools in Caenorhabditis elegans.

red PhyB/PIF3 C. elegans in vivo Immediate control of second messengers
ACS Synth Biol, 20 Feb 2023 DOI: 10.1021/acssynbio.2c00461 Link to full text
Abstract: 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.

Coupling Cell Communication and Optogenetics: Implementation of a Light-Inducible Intercellular System in Yeast.

blue VVD S. cerevisiae Signaling cascade control Transgene expression
ACS Synth Biol, 19 Dec 2022 DOI: 10.1021/acssynbio.2c00338 Link to full text
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.

Upconversion Optogenetic Engineered Bacteria System for Time-Resolved Imaging Diagnosis and Light-Controlled Cancer Therapy.

blue YtvA E. coli Transgene expression Cell death
ACS Appl Mater Interfaces, 6 Oct 2022 DOI: 10.1021/acsami.2c14633 Link to full text
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.

Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

blue AsLOV2 HEK293 Transgene expression
ACS Synth Biol, 30 Sep 2022 DOI: 10.1021/acssynbio.2c00232 Link to full text
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.

Light-Dependent Control of Bacterial Expression at the mRNA Level.

blue PAL YtvA E. coli Transgene expression
ACS Synth Biol, 21 Sep 2022 DOI: 10.1021/acssynbio.2c00365 Link to full text
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.

Optogenetic Control of Bacterial Expression by Red Light.

blue red DrBphP PAL E. coli Transgene expression
ACS Synth Biol, 23 Aug 2022 DOI: 10.1021/acssynbio.2c00259 Link to full text
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.

Implementation of a Novel Optogenetic Tool in Mammalian Cells Based on a Split T7 RNA Polymerase.

blue Magnets VVD HEK293T Transgene expression
ACS Synth Biol, 3 Aug 2022 DOI: 10.1021/acssynbio.2c00067 Link to full text
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.

Light-Sensitive Lactococcus lactis for Microbe-Gut-Brain Axis Regulating via Upconversion Optogenetic Micro-Nano System.

blue YtvA L. lactis Transgene expression
ACS Nano, 1 Apr 2022 DOI: 10.1021/acsnano.1c11536 Link to full text
Abstract: The discovery of the gut-brain axis has proven that brain functions can be affected by the gut microbiota's metabolites, so there are significant opportunities to explore new tools to regulate gut microbiota and thus work on the brain functions. Meanwhile, engineered bacteria as oral live biotherapeutic agents to regulate the host's healthy homeostasis have attracted much attention in microbial therapy. However, whether this strategy is able to remotely regulate the host's brain function in vivo has not been investigated. Here, we engineered three blue-light-responsive probiotics as oral live biotherapeutic agents. They are spatiotemporally delivered and controlled by the upconversion optogenetic micro-nano system. This micro-nano system promotes the small intestine targeting and production of the exogenous L. lactis in the intestines, which realizes precise manipulation of brain functions including anxiety behavior, Parkinson's disease, and vagal afferent. The noninvasive and real-time probiotic intervention strategy makes the communiation from the gut to the host more controllable, which will enable the potential for engineered microbes accurately and effectively regulating a host's health.

NIR-Responsive Photodynamic Nanosystem Combined with Antitumor Immune Optogenetics Bacteria for Precise Synergetic Therapy.

blue YtvA L. lactis Transgene expression
ACS Appl Mater Interfaces, 9 Mar 2022 DOI: 10.1021/acsami.2c01138 Link to full text
Abstract: Photodynamic therapy (PDT) and immunotherapy are considered promising methods for the treatment of tumors. However, these treatment systems are still suffering from shortcomings such as hypoxia, easy metastasis, and delayed immune response during PDT. Therefore, it is still challenging to establish a programmed and rapid response immune combination therapy platform. Here, we construct a two-step synergetic therapy platform for the treatment of primary tumors and distant tumors using upconversion nanoparticles (UCNPs) and engineered bacteria as therapeutic media. In the first step, erbium ion (Er3+)-doped UCNPs act as a photoswitcher to activate the photosensitizer ZnPc to produce 1O2 for primary tumor therapy. In the second step, thulium ion (Tm3+)-doped UCNPs can emit blue-violet light under the excitation of near-infrared (NIR) light to activate the engineered bacteria to produce interferon (INF-γ) and release them in the intestine, which can not only treat tumors directly but also act with PDT to regulate immune pathways to activate the immune system, resulting in a joint immunotherapy effect to inhibit the growth of distant tumors. As a new type of programmatic combination therapy, we have proved that this platform can jointly activate the body's immune system during PDT and immunization treatment and can effectively inhibit tumor metastasis.

Systematic In Vivo Characterization of Fluorescent Protein Maturation in Budding Yeast.

blue EL222 S. cerevisiae Transgene expression
ACS Synth Biol, 18 Feb 2022 DOI: 10.1021/acssynbio.1c00387 Link to full text
Abstract: Fluorescent protein (FP) maturation can limit the accuracy with which dynamic intracellular processes are captured and reduce the in vivo brightness of a given FP in fast-dividing cells. The knowledge of maturation timescales can therefore help users determine the appropriate FP for each application. However, in vivo maturation rates can greatly deviate from in vitro estimates that are mostly available. In this work, we present the first systematic study of in vivo maturation for 12 FPs in budding yeast. To overcome the technical limitations of translation inhibitors commonly used to study FP maturation, we implemented a new approach based on the optogenetic stimulations of FP expression in cells grown under constant nutrient conditions. Combining the rapid and orthogonal induction of FP transcription with a mathematical model of expression and maturation allowed us to accurately estimate maturation rates from microscopy data in a minimally invasive manner. Besides providing a useful resource for the budding yeast community, we present a new joint experimental and computational approach for characterizing FP maturation, which is applicable to a wide range of organisms.

Far-Red Light Triggered Production of Bispecific T Cell Engagers (BiTEs) from Engineered Cells for Antitumor Application.

red BphS HEK293T Hep G2 SK-HEP-1 Transgene expression
ACS Synth Biol, 3 Feb 2022 DOI: 10.1021/acssynbio.1c00523 Link to full text
Abstract: Bispecific T-cell engagers (BiTEs), which have shown potent antitumor activity in humans, are emerging as one of the most promising immunotherapeutic strategies for cancer treatment in recent years. However, the clinical application of BiTEs nowadays has been hampered by their short half-life in the circulatory system due to their low molecular weight and rapid renal clearance. Inevitable continuous infusion of BiTEs has become a routine operation in order to achieve effective treatment, although it is costly, inconvenient, time-consuming, and even painful for patients in some cases. To develop an on-demand, tunable, and reversible approach to overcome these limitations, we assembled a transcription-control device into mammalian cells based on a bacterial far-red light (FRL) responsive signaling pathway to drive the expression of a BiTE against Glypican 3 (GPC3), which is a highly tumor-specific antigen expressed in most hepatocellular carcinomas (HCC). As demonstrated in in vitro experiments, we proved that the FRL sensitive device spatiotemporally responded to the control of FRL illumination and produced a therapeutic level of BiTEs that recruited and activated human T cells to eliminate GPC3 positive tumor cells. By functionally harnessing the power of optogenetics to remotely regulate the production of BiTEs from bioengineered cells and demonstrating its effectiveness in treating tumor cells, this study provides a novel approach to achieve an in vivo supply of BiTEs, which could be potentially applied to other formats of bispecific antibodies and facilitate their clinical applications.

Design and Characterization of an Optogenetic System in Pichia pastoris.

blue EL222 P. pastoris Transgene expression
ACS Synth Biol, 7 Jan 2022 DOI: 10.1021/acssynbio.1c00422 Link to full text
Abstract: Pichia pastoris (P. pastoris) is the workhorse in the commercial production of many valuable proteins. Traditionally, the regulation of gene expression in P. pastoris is achieved through induction by methanol which is toxic and flammable. The emerging optogenetic technology provides an alternative and cleaner gene regulation method. Based on the photosensitive protein EL222, we designed a novel "one-component" optogenetic system. The highest induction ratio was 79.7-fold under blue light compared to the group under darkness. After switching cells from dark to blue illumination, the system induced expression in just 1 h. Only 2 h after the system was switched back to the darkness from blue illumination, the target gene expression was inactivated 5-fold. The induction intensity of the optogenetic system is positively correlated with the dose and periodicity of blue illumination, and it has good spatial control. These results provide the first credible case of optogenetically induced protein expression in P. pastoris.

Designing Single-Component Optogenetic Membrane Recruitment Systems: The Rho-Family GTPase Signaling Toolbox.

blue BcLOV4 HEK293T Signaling cascade control
ACS Synth Biol, 3 Jan 2022 DOI: 10.1021/acssynbio.1c00604 Link to full text
Abstract: We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.

Analysis of Three Architectures for Controlling PTP1B with Light.

blue AsLOV2 LOVTRAP Cos-7 E. coli HEK293T Transgene expression
ACS Synth Biol, 13 Dec 2021 DOI: 10.1021/acssynbio.1c00398 Link to full text
Abstract: Photosensory domains are powerful tools for placing proteins under optical control, but their integration into light-sensitive chimeras is often challenging. Many designs require structural iterations, and direct comparisons of alternative approaches are rare. This study uses protein tyrosine phosphatase 1B (PTP1B), an influential regulatory enzyme, to compare three architectures for controlling PTPs with light: a protein fusion, an insertion chimera, and a split construct. All three designs permitted optical control of PTP1B activity in vitro (i.e., kinetic assays of purified enzyme) and in mammalian cells; photoresponses measured under both conditions, while different in magnitude, were linearly correlated. The fusion- and insertion-based architectures exhibited the highest dynamic range and maintained native localization patterns in mammalian cells. A single insertion architecture enabled optical control of both PTP1B and TCPTP, but not SHP2, where the analogous chimera was active but not photoswitchable. Findings suggest that PTPs are highly tolerant of domain insertions and support the use of in vitro screens to evaluate different optogenetic designs.

An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance.

blue iLID LOVTRAP in vitro S. cerevisiae Transgene expression
ACS Synth Biol, 19 Nov 2021 DOI: 10.1021/acssynbio.1c00350 Link to full text
Abstract: Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

Exploiting Noise, Non-Linearity, and Feedback for Differential Control of Multiple Synthetic Cells with a Single Optogenetic Input.

blue Magnets in silico
ACS Synth Biol, 18 Nov 2021 DOI: 10.1021/acssynbio.1c00341 Link to full text
Abstract: Synthetic biology seeks to develop modular biocircuits that combine to produce complex, controllable behaviors. These designs are often subject to noisy fluctuations and uncertainties, and most modern synthetic biology design processes have focused to create robust components to mitigate the noise of gene expression and reduce the heterogeneity of single-cell responses. However, a deeper understanding of noise can achieve control goals that would otherwise be impossible. We explore how an "Optogenetic Maxwell Demon" could selectively amplify noise to control multiple cells using single-input-multiple-output (SIMO) feedback. Using data-constrained stochastic model simulations and theory, we show how an appropriately selected stochastic SIMO controller can drive multiple different cells to different user-specified configurations irrespective of initial conditions. We explore how controllability depends on cells' regulatory structures, the amount of information available to the controller, and the accuracy of the model used. Our results suggest that gene regulation noise, when combined with optogenetic feedback and non-linear biochemical auto-regulation, can achieve synergy to enable precise control of complex stochastic processes.

Light-Responsive Dynamic Protein Hydrogels Based on LOVTRAP.

blue LOVTRAP in vitro Extracellular optogenetics
Langmuir, 15 Aug 2021 DOI: 10.1021/acs.langmuir.1c01699 Link to full text
Abstract: Protein-based hydrogels can mimic many aspects of native extracellular matrices (ECMs) and are promising biomedical materials that find various applications in cell proliferation, drug/cell delivery, and tissue engineering. To be adapted for different tasks, it is important that the mechanical and/or biochemical properties of protein-based hydrogels can be regulated by external stimuli. Light as a regulation stimulus is of advantage because it can be easily applied in demanded spatiotemporal manners. The noncovalent binding between the light-oxygen-voltage-sensing domain 2 (LOV2) and its binding partner ZDark1 (zdk1), named as LOVTRAP, is a light-responsive interaction. The binding affinity of LOVTRAP is much higher in dark than that under blue light irradiation. Taking advantage of these light-responsive interactions, herein we endeavored to use LOVTRAP as a crosslinking mechanism to engineer light-responsive protein hydrogels. Using LOV2-containing and zdk1-containing multifunctional protein building blocks, we successfully engineered a light-responsive protein hydrogel whose viscoelastic properties can change in response to light: in the dark, the hydrogel showed higher storage modulus; under blue light irradiation, the storage modulus decreased. Due to the noncovalent nature of the LOVTRAP, the engineered LOVTRAP protein hydrogels displayed shear-thinning and self-healing properties and served as an excellent injectable protein hydrogel. We anticipated that this new class of light-responsive protein hydrogels will broaden the scope of dynamic protein hydrogels and help develop other light-responsive protein hydrogels for biomedical applications.

Optogenetic Control of Microbial Consortia Populations for Chemical Production.

blue YtvA E. coli S. cerevisiae Transgene expression
ACS Synth Biol, 5 Aug 2021 DOI: 10.1021/acssynbio.1c00182 Link to full text
Abstract: Microbial co-culture fermentations can improve chemical production from complex biosynthetic pathways over monocultures by distributing enzymes across multiple strains, thereby reducing metabolic burden, overcoming endogenous regulatory mechanisms, or exploiting natural traits of different microbial species. However, stabilizing and optimizing microbial subpopulations for maximal chemical production remains a major obstacle in the field. In this study, we demonstrate that optogenetics is an effective strategy to dynamically control populations in microbial co-cultures. Using a new optogenetic circuit we call OptoTA, we regulate an endogenous toxin-antitoxin system, enabling tunability of Escherichia coli growth using only blue light. With this system we can control the population composition of co-cultures of E. coli and Saccharomyces cerevisiae. When introducing in each strain different metabolic modules of biosynthetic pathways for isobutyl acetate or naringenin, we found that the productivity of co-cultures increases by adjusting the population ratios with specific light duty cycles. This study shows the feasibility of using optogenetics to control microbial consortia populations and the advantages of using light to control their chemical production.

The Neurospora crassa Inducible Q System Enables Simultaneous Optogenetic Amplification and Inversion in Saccharomyces cerevisiae for Bidirectional Control of Gene Expression.

blue EL222 S. cerevisiae Transgene expression
ACS Synth Biol, 4 Aug 2021 DOI: 10.1021/acssynbio.1c00229 Link to full text
Abstract: Bidirectional optogenetic control of yeast gene expression has great potential for biotechnological applications. Our group has developed optogenetic inverter circuits that activate transcription using darkness, as well as amplifier circuits that reach high expression levels under limited light. However, because both types of circuits harness Gal4p and Gal80p from the galactose (GAL) regulon they cannot be used simultaneously. Here, we apply the Q System, a transcriptional activator/inhibitor system from Neurospora crassa, to build circuits in Saccharomyces cerevisiae that are inducible using quinic acid, darkness, or blue light. We develop light-repressed OptoQ-INVRT circuits that initiate darkness-triggered transcription within an hour of induction, as well as light-activated OptoQ-AMP circuits that achieve up to 39-fold induction. The Q System does not exhibit crosstalk with the GAL regulon, allowing coutilization of OptoQ-AMP circuits with previously developed OptoINVRT circuits. As a demonstration of practical applications in metabolic engineering, we show how simultaneous use of these circuits can be used to dynamically control both growth and production to improve acetoin production, as well as enable light-tunable co-production of geraniol and linalool, two terpenoids implicated in the hoppy flavor of beer. OptoQ-AMP and OptoQ-INVRT circuits enable simultaneous optogenetic signal amplification and inversion, providing powerful additions to the yeast optogenetic toolkit.
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