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 163 results

Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation.

blue cpLOV2 HEK293 mouse in vivo primary mouse cortical neurons Transgene expression
bioRxiv, 31 Aug 2023 DOI: 10.1101/2023.08.30.554643 Link to full text
Abstract: The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is one of the primary triggers for initiating cap-dependent translation. Amongst its functions, mTORC1 phosphorylates eIF4E-binding proteins (4E-BPs), which prevents them from binding to eIF4E and thereby enables translation initiation. mTORC1 signaling is required for multiple forms of protein synthesis- dependent synaptic plasticity and various forms of long-term memory (LTM), including associative threat memory. However, the approaches used thus far to target mTORC1 and its effectors, such as pharmacological inhibitors or genetic knockouts, lack fine spatial and temporal control. The development of a conditional and inducible eIF4E knockdown mouse line partially solved the issue of spatial control, but still lacked optimal temporal control to study memory consolidation. Here, we have designed a novel optogenetic tool (Opto4E-BP) for cell type-specific, light-dependent regulation of eIF4E in the brain. We show that light-activation of Opto4E-BP decreases protein synthesis in HEK cells and primary mouse neurons. In situ, light-activation of Opto4E-BP in excitatory neurons decreased protein synthesis in acute amygdala slices. Finally, light activation of Opto4E-BP in principal excitatory neurons in the lateral amygdala (LA) of mice after training blocked the consolidation of LTM. The development of this novel optogenetic tool to modulate eIF4E-dependent translation with spatiotemporal precision will permit future studies to unravel the complex relationship between protein synthesis and the consolidation of LTM.

C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways.

blue iLID S. cerevisiae Transgene expression
J Biol Chem, 16 Aug 2023 DOI: 10.1016/j.jbc.2023.105166 Link to full text
Abstract: Protein quality control (PQC) mechanisms are essential for degradation of misfolded or dysfunctional proteins. An essential part of protein homeostasis is recognition of defective proteins by PQC components and their elimination by the ubiquitin-proteasome system, often concentrating on protein termini as indicators of protein integrity. Changes in amino acid composition of C-terminal ends arise through protein disintegration, alternative splicing or during the translation step of protein synthesis from premature termination or translational stop-codon read-through. We characterized reporter protein stability using light-controlled exposure of random C-terminal peptides (CtPC) in budding yeast revealing stabilizing and destabilizing features of amino acids at positions -5 to -1 of the C-terminus. The (de)stabilization properties of CtPC-degrons depend on amino acid identity, position as well as composition of the C-terminal sequence and are transferable. Evolutionary pressure towards stable proteins in yeast is evidenced by amino acid residues under-represented in cytosolic and nuclear proteins at corresponding C-terminal positions, but over-represented in unstable CtPC-degrons, and vice versa. Furthermore, analysis of translational stop-codon read-through peptides suggested that such extended proteins have destabilizing C-termini. PQC pathways targeting CtPC-degrons involved the ubiquitin-protein ligase Doa10 and the cullin-RING E3 ligase (CRL) SCFDas1. Overall, our data suggest a proteome protection mechanism that targets proteins with unnatural C-termini by recognizing a surprisingly large number of C-terminal sequence variants.

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.

Multidimensional characterization of inducible promoters and a highly light-sensitive LOV-transcription factor.

blue red EL222 PhyB/PIF3 S. cerevisiae Transgene expression
Nat Commun, 27 Jun 2023 DOI: 10.1038/s41467-023-38959-8 Link to full text
Abstract: The ability to independently control the expression of different genes is important for quantitative biology. Using budding yeast, we characterize GAL1pr, GALL, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr, Z3EV, blue-light inducible optogenetic systems El222-LIP, El222-GLIP, and red-light inducible PhyB-PIF3. We report kinetic parameters, noise scaling, impact on growth, and the fundamental leakiness of each system using an intuitive unit, maxGAL1. We uncover disadvantages of widely used tools, e.g., nonmonotonic activity of MET3pr and GALL, slow off kinetics of the doxycycline- and estradiol-inducible systems tetOpr and Z3EV, and high variability of PHO5pr and red-light activated PhyB-PIF3 system. We introduce two previously uncharacterized systems: strongLOV, a more light-sensitive El222 mutant, and ARG3pr, which is induced in the absence of arginine or presence of methionine. To demonstrate fine control over gene circuits, we experimentally tune the time between cell cycle Start and mitosis, artificially simulating near-wild-type timing. All strains, constructs, code, and data ( ) are made available.

A Bioluminescent Activity Dependent (BLADe) Platform for Converting Neuronal Activity to Photoreceptor Activation.

blue EL222 HEK293 HeLa mouse in vivo Transgene expression
bioRxiv, 27 Jun 2023 DOI: 10.1101/2023.06.25.546469 Link to full text
Abstract: We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.

Detecting Photoactivatable Cre-mediated Gene Deletion Efficiency in Escherichia coli.

blue Magnets E. coli Transgene expression
Bio Protoc, 5 Jun 2023 DOI: 10.21769/bioprotoc.4685 Link to full text
Abstract: Gene deletion is one of the standard approaches in genetics to investigate the roles and functions of target genes. However, the influence of gene deletion on cellular phenotypes is usually analyzed sometime after the gene deletion was introduced. Such lags from gene deletion to phenotype evaluation could select only the fittest fraction of gene-deleted cells and hinder the detection of potentially diverse phenotypic consequences. Therefore, dynamic aspects of gene deletion, such as real-time propagation and compensation of deletion effects on cellular phenotypes, still need to be explored. To resolve this issue, we have recently introduced a new method that combines a photoactivatable Cre recombination system and microfluidic single-cell observation. This method enables us to induce gene deletion at desired timings in single bacterial cells and to monitor their dynamics for prolonged periods. Here, we detail the protocol for estimating the fractions of gene-deleted cells based on a batch-culture assay. The duration of blue light exposure significantly affects the fractions of gene-deleted cells. Therefore, gene-deleted and non-deleted cells can coexist in a cellular population by adjusting the duration of blue light exposure. Single-cell observations under such illumination conditions allow the comparison of temporal dynamics between gene-deleted and non-deleted cells and unravel phenotypic dynamics provoked by gene deletion.

Light-switchable transcription factors obtained by direct screening in mammalian cells.

blue AsLOV2 HEK293T Transgene expression
Nat Commun, 2 Jun 2023 DOI: 10.1038/s41467-023-38993-6 Link to full text
Abstract: Optogenetic tools can provide fine spatial and temporal control over many biological processes. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. We demonstrate the approach's utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression.

blue VVD HEK293T Transgene expression
Int J Mol Sci, 31 May 2023 DOI: 10.3390/ijms24119537 Link to full text
Abstract: Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

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.

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

blue AsLOV2 iLID E. coli HEK293T mouse in vivo rat cortical neurons S. cerevisiae Transgene expression
Nat Methods, 15 May 2023 DOI: 10.1038/s41592-023-01880-5 Link to full text
Abstract: The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

Optogenetic spatial patterning of cooperation in yeast populations.

blue EL222 S. cerevisiae Transgene expression
bioRxiv, 15 May 2023 DOI: 10.1101/2023.05.15.540783 Link to full text
Abstract: Microbial communities are a siege of complex metabolic interactions such as cooperation and competition for resources. Methods to control such interactions could lead to major advances in our ability to engineer microbial consortia for bioproduction and synthetic biology applications. Here, we used optogenetics to control invertase production in yeast, thereby creating landscapes of cooperator and cheater cells. Yeast cells behave as cooperators (i.e., transform sucrose into glucose, a public “good”) upon blue light illumination or cheaters (i.e., consume glucose produced by cooperators to grow) in the dark. We show that cooperators benefit best from the hexoses they produce when their domain size is constrained between two cut-off length-scales. From an engineering point of view, the system behaves as a band pass filter. The lower limit is the trace of cheaters’ competition for hexoses, while the upper limit is defined by cooperators’ competition for sucrose. Hence, cooperation mostly occurs at the frontiers with cheater cells, which not only compete for hexoses but also cooperate passively by letting sucrose reach cooperators. We anticipate that this optogenetic method could be applied to shape metabolic interactions in a variety of microbial ecosystems.

Light-Regulated Pro-Angiogenic Engineered Living Materials.

blue YtvA E. coli Transgene expression
Adv Funct Mater, 5 May 2023 DOI: 10.1002/adfm.202212695 Link to full text
Abstract: Regenerative medicine aims to restore damaged cells, tissues, and organs, for which growth factors are vital to stimulate regenerative cellular transformations. Major advances have been made in growth factor engineering and delivery like the development of robust peptidomimetics and controlled release matrices. However, their clinical applicability remains limited due to their poor stability in the body and need for careful regulation of their local concentration to avoid unwanted side-effects. In this study, a strategy to overcome these limitations is explored using engineered living materials (ELMs), which contain live microorganisms that can be programmed with stimuli-responsive functionalities. Specifically, the development of an ELM that releases a pro-angiogenic protein in a light-regulated manner is described. This is achieved by optogenetically engineering bacteria to synthesize and secrete a vascular endothelial growth factor peptidomimetic (QK) linked to a collagen-binding domain. The bacteria are securely encapsulated in bilayer hydrogel constructs that support bacterial functionality but prevent their escape from the ELM. In situ control over the release profiles of the pro-angiogenic protein using light is demonstrated. Finally, it is shown that the released protein is able to bind collagen and promote angiogenic network formation among vascular endothelial cells, indicating the regenerative potential of these ELMs.

Transcription factor localization dynamics and DNA binding drive distinct promoter interpretations.

blue LOVTRAP S. cerevisiae Transgene expression
Cell Rep, 21 Apr 2023 DOI: 10.1016/j.celrep.2023.112426 Link to full text
Abstract: Environmental information may be encoded in the temporal dynamics of transcription factor (TF) activation and subsequently decoded by gene promoters to enact stimulus-specific gene expression programs. Previous studies of this behavior focused on the encoding and decoding of information in TF nuclear localization dynamics, yet cells control the activity of TFs in myriad ways, including by regulating their ability to bind DNA. Here, we use light-controlled mutants of the yeast TF Msn2 as a model system to investigate how promoter decoding of TF localization dynamics is affected by changes in the ability of the TF to bind DNA. We find that yeast promoters directly decode the light-controlled localization dynamics of Msn2 and that the effects of changing Msn2 affinity on that decoding behavior are highly promoter dependent, illustrating how cells could regulate TF localization dynamics and DNA binding in concert for improved control of gene expression.

Requirements for mammalian promoters to decode transcription factor dynamics.

blue AsLOV2 HEK293 HeLa Transgene expression Endogenous gene expression
Nucleic Acids Res, 18 Apr 2023 DOI: 10.1093/nar/gkad273 Link to full text
Abstract: In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

Light-activated macromolecular phase separation modulates transcription by reconfiguring chromatin interactions.

blue CRY2clust HeLa Transgene expression Endogenous gene expression
Sci Adv, 31 Mar 2023 DOI: 10.1126/sciadv.adg1123 Link to full text
Abstract: Biomolecular condensates participate in the regulation of gene transcription, yet the relationship between nuclear condensation and transcriptional activation remains elusive. Here, we devised a biotinylated CRISPR-dCas9-based optogenetic method, light-activated macromolecular phase separation (LAMPS), to enable inducible formation, affinity purification, and multiomic dissection of nuclear condensates at the targeted genomic loci. LAMPS-induced condensation at enhancers and promoters activates endogenous gene transcription by chromatin reconfiguration, causing increased chromatin accessibility and de novo formation of long-range chromosomal loops. Proteomic profiling of light-induced condensates by dCas9-mediated affinity purification uncovers multivalent interaction-dependent remodeling of macromolecular composition, resulting in the selective enrichment of transcriptional coactivators and chromatin structure proteins. Our findings support a model whereby the formation of nuclear condensates at native genomic loci reconfigures chromatin architecture and multiprotein assemblies to modulate gene transcription. Hence, LAMPS facilitates mechanistic interrogation of the relationship between nuclear condensation, genome structure, and gene transcription in living cells.

Optogenetic closed-loop feedback control of the unfolded protein response optimizes protein production.

blue EL222 S. cerevisiae Transgene expression
Metab Eng, 11 Mar 2023 DOI: 10.1016/j.ymben.2023.03.001 Link to full text
Abstract: In biotechnological protein production processes, the onset of protein unfolding at high gene expression levels leads to diminishing production yields and reduced efficiency. Here we show that in silico closed-loop optogenetic feedback control of the unfolded protein response (UPR) in S. cerevisiae clamps gene expression rates at intermediate near-optimal values, leading to significantly improved product titers. Specifically, in a fully-automated custom-built 1L-photobioreactor, we used a cybergenetic control system to steer the level of UPR in yeast to a desired set-point by optogenetically modulating the expression of α-amylase, a hard-to-fold protein, based on real-time feedback measurements of the UPR, resulting in 60% higher product titers. This proof-of-concept study paves the way for advanced optimal biotechnology production strategies that diverge from and complement current strategies employing constitutive overexpression or genetically hardwired circuits.

Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.

blue CRY2/CRY2 A-431 Organelle manipulation Transgene expression
Cell Rep, 11 Mar 2023 DOI: 10.1016/j.celrep.2023.112229 Link to full text
Abstract: Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.

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.

Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s.

blue green red Am1 c0023g2/BAm green Am1 c0023g2/BAm red AsLOV2 TULIP CHO-K1 HEK293T in vitro S. cerevisiae Transgene expression Multichromatic
Nat Methods, 23 Feb 2023 DOI: 10.1038/s41592-023-01764-8 Link to full text
Abstract: Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

An optogenetic toolkit for light-inducible antibiotic resistance.

blue VVD E. coli Transgene expression Nucleic acid editing
Nat Commun, 23 Feb 2023 DOI: 10.1038/s41467-023-36670-2 Link to full text
Abstract: Antibiotics are a key control mechanism for synthetic biology and microbiology. Resistance genes are used to select desired cells and regulate bacterial populations, however their use to-date has been largely static. Precise spatiotemporal control of antibiotic resistance could enable a wide variety of applications that require dynamic control of susceptibility and survival. Here, we use light-inducible Cre recombinase to activate expression of drug resistance genes in Escherichia coli. We demonstrate light-activated resistance to four antibiotics: carbenicillin, kanamycin, chloramphenicol, and tetracycline. Cells exposed to blue light survive in the presence of lethal antibiotic concentrations, while those kept in the dark do not. To optimize resistance induction, we vary promoter, ribosome binding site, and enzyme variant strength using chromosome and plasmid-based constructs. We then link inducible resistance to expression of a heterologous fatty acid enzyme to increase production of octanoic acid. These optogenetic resistance tools pave the way for spatiotemporal control of cell survival.

Spatiotemporally controllable diphtherin transgene system and neoantigen immunotherapy.

blue VVD B16-F10 mouse in vivo Transgene expression
J Control Release, 14 Feb 2023 DOI: 10.1016/j.jconrel.2022.08.059 Link to full text
Abstract: Individualized immunotherapy has attracted great attention due to its high specificity, effectiveness, and safety. We used an exogenous antigen to label tumor cells with MHC I molecules, which allowed neoantigen-specific T cells to recognize and kill tumor cells. A neoantigen vaccine alone cannot achieve complete tumor clearance due to a tumor immunosuppressive microenvironment. The LightOn system was developed to effectively eliminate tumor cells through the spatiotemporally controllable expression of diphtheria toxin A fragment, leading to antigen release in the tumor region. These antigens stimulated and enhanced immunological function and thus, recruited neoantigen-specific T cells to infiltrate tumor tissue. Using the nanoparticle delivery system, neoantigens produced higher delivery efficiency to lymph nodes and improved tumor targeting ability for tumor cell labelling. Good tumor inhibition and prolonged survival were achieved, while eliciting a strong immune response. The combination of a spatiotemporally controllable transgene system with tumor neoantigen labeling has great potential for tumor immunotherapy.

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales.

blue EL222 S. cerevisiae Transgene expression
Front Bioeng Biotechnol, 6 Feb 2023 DOI: 10.3389/fbioe.2023.1085268 Link to full text
Abstract: Optogenetics arises as a valuable tool to precisely control genetic circuits in microbial cell factories. Light control holds the promise of optimizing bioproduction methods and maximizing yields, but its implementation at different steps of the strain development process and at different culture scales remains challenging. In this study, we aim to control beta-carotene bioproduction using optogenetics in Saccharomyces cerevisiae and investigate how its performance translates across culture scales. We built four lab-scale illumination devices, each handling different culture volumes, and each having specific illumination characteristics and cultivating conditions. We evaluated optogenetic activation and beta-carotene production across devices and optimized them both independently. Then, we combined optogenetic induction and beta-carotene production to make a light-inducible beta-carotene producer strain. This was achieved by placing the transcription of the bifunctional lycopene cyclase/phytoene synthase CrtYB under the control of the pC120 optogenetic promoter regulated by the EL222-VP16 light-activated transcription factor, while other carotenogenic enzymes (CrtI, CrtE, tHMG) were expressed constitutively. We show that illumination, culture volume and shaking impact differently optogenetic activation and beta-carotene production across devices. This enabled us to determine the best culture conditions to maximize light-induced beta-carotene production in each of the devices. Our study exemplifies the stakes of scaling up optogenetics in devices of different lab scales and sheds light on the interplays and potential conflicts between optogenetic control and metabolic pathway efficiency. As a general principle, we propose that it is important to first optimize both components of the system independently, before combining them into optogenetic producing strains to avoid extensive troubleshooting. We anticipate that our results can help designing both strains and devices that could eventually lead to larger scale systems in an effort to bring optogenetics to the industrial scale.

Polarized branched Actin modulates cortical mechanics to produce unequal-size daughters during asymmetric division.

blue CRY2/CIB1 TULIP D. melanogaster in vivo Cell cycle control Transgene expression
Nat Cell Biol, 6 Feb 2023 DOI: 10.1038/s41556-022-01058-9 Link to full text
Abstract: The control of cell shape during cytokinesis requires a precise regulation of mechanical properties of the cell cortex. Only few studies have addressed the mechanisms underlying the robust production of unequal-sized daughters during asymmetric cell division. Here we report that unequal daughter-cell sizes resulting from asymmetric sensory organ precursor divisions in Drosophila are controlled by the relative amount of cortical branched Actin between the two cell poles. We demonstrate this by mistargeting the machinery for branched Actin dynamics using nanobodies and optogenetics. We can thereby engineer the cell shape with temporal precision and thus the daughter-cell size at different stages of cytokinesis. Most strikingly, inverting cortical Actin asymmetry causes an inversion of daughter-cell sizes. Our findings uncover the physical mechanism by which the sensory organ precursor mother cell controls relative daughter-cell size: polarized cortical Actin modulates the cortical bending rigidity to set the cell surface curvature, stabilize the division and ultimately lead to unequal daughter-cell size.

A red light-controlled probiotic bio-system for in-situ gut-brain axis regulation.

red Cph1 E. coli Transgene expression Cell death
Biomaterials, 20 Jan 2023 DOI: 10.1016/j.biomaterials.2023.122005 Link to full text
Abstract: Microbes regulate brain function through the gut-brain axis, deriving the technology to modulate the gut-brain axis in situ by engineered probiotics. Optogenetics offers precise and flexible strategies for controlling the functions of probiotics in situ. However, the poor penetration of most frequently used short wavelength light has limited the application of optogenetic probiotics in the gut. Herein, a red-light optogenetic gut probiotic was applied for drug production and delivery and regulation of the host behaviors. Firstly, a Red-light Optogenetic E. coli Nissle 1917 strain (ROEN) that could respond to red light and release drug product by light-controlled lysis was constructed. The remaining optical power of red light after 3 cm tissue was still able to initiate gene expression of ROEN and produce about approximately 3-fold induction efficiency. To give full play to the in vivo potential of ROEN, its responsive ability of the penetrated red light was tested, and its encapsulation was realized by PH-sensitive alginate microcapsules for further oral administration. The function of ROEN for gut-brain regulation was realized by releasing Exendin-4 fused with anti-neonatal Fc receptor affibody. Neuroprotection and behavioral regulation effects were evaluated in the Parkinson's disease mouse model, after orally administration of ROEN delivering Exendin-4 under optogenetic control in the murine gut. The red-light optogenetic probiotic might be a perspective platform for in situ drug delivery and gut-brain axis regulation.

Generation of a photocontrollable recombinant bovine parainfluenza virus type 3.

blue Magnets HEK293T MDBK Transgene expression
Microbiol Immunol, 6 Jan 2023 DOI: 10.1111/1348-0421.13052 Link to full text
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.
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