Qr: application:" Transgene expression"
Showing 1 - 25 of 243 results
1.
Engineering an Optogenetic pH-Modulator in Bacteria.
Abstract:
Cells in many naturally occurring organisms routinely cooperate to control their extracellular pH in a dynamic and reversible manner, but this capability has been underexplored in synthetic biology. Here, we sought to engineer a microbial system that switches between two states -high and low extracellular pH- with minimal human intervention. We accomplished this by combining: (1) a genetic circuit that produces recombinant urease under the control of a light-inducible promoter; (2) a degradation tag on urease to accelerate the high-to-low pH transition; and (3) optimization of several environmental factors, including media composition, replenishment rate, and light exposure patterns. The system raises the pH when urease is produced and hydrolyzes urea in the media to produce ammonia; it lowers the pH as a byproduct of the cell's native metabolism when urease production ceases. We demonstrate that the optimized system cycles continuously for up to 14 days with minimal performance loss. Overall, our system demonstrates synthetic pH control in an engineered living system and highlights challenges and potential solutions for using such systems outside of the context of typical laboratory manipulation.
2.
Phage-assisted evolution of allosteric protein switches.
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Southern, NT
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von Bachmann, A
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Hovsepyan, A
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Griebl, M
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Wolf, B
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Lemmen, N
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Kroell, AS
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Westermann, S
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Mathony, J
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Niopek, D
Abstract:
Allostery, the transmission of locally induced conformational changes to distant functional sites, is a key mechanism for protein regulation. Artificial allosteric effectors enable remote manipulation of cell function; their engineering, however, is hampered by our limited understanding of allosteric residue networks. Here, we introduce a phage-assisted evolution platform for in vivo optimization of allosteric proteins. It applies opposing selection pressures to enhance activity and switchability of phage-encoded effectors and leverages retron-based recombineering to broadly explore fitness landscapes, introducing point mutations, insertions, and deletions. Applying this framework to the transcription factor AraC yielded near-binary optogenetic switches, with light-controlled activity spanning ~1000-fold dynamic range. Long-read sequencing across selection cycles enabled high-resolution tracking of evolving variant pools, revealing adaptive trajectories and context-dependent residue interactions. Mechanistically, we find that linker mutations promoting α-helix extension at the sensor-effector junction enhance conformational coupling between LOV2 and AraC. These variants emerge consistently across independently evolved pools, underscoring their functional relevance. Together, we develop a framework for the directed evolution of programmable allosteric switches in vivo. By coupling dynamic selection with deep mutational scanning and temporal sequencing, it enables both functional optimization and mechanistic insight into allosteric networks.
3.
Enhancing the performance of Magnets photosensors.
Abstract:
Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Tailoring their properties enables their full exploitation for optogenetic regulation in basic research and applied bioengineering applications. Here, we present a simple, yet powerful strategy based on random mutagenesis coupled to high-throughput screening that allowed altering the most fundamental properties of the widely used nMag/pMag photodimerization system: its light sensitivity and activation. Variants were characterized in vivo in bacteria by flow cytometry and during the entire growth curve by spectrofluorometry. We identify mutations that either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the light activation and dark-to-light fold change. Notably, light sensitivity and activation levels could be changed independently. In addition, we demonstrated that the shapes of the dose-response curves can be finely tuned. This broadens the applicability of the Magnets photosensors for optogenetic regulation strategies.
4.
Light-directed evolution of dynamic, multi-state, and computational protein functionalities.
Abstract:
Evolving dynamic, multi-state, and computational protein functionalities is challenging because it requires selection pressure on all the states of a protein of interest (POI) and the transitions between them. To create a continuous directed evolution paradigm for such properties, we genetically engineered budding yeast for optogenetic input to switch a POI "on" and "off," which, in turn, controls a Cdk1 cyclin that is essential for one cell-cycle stage but detrimental for another. The method, "optovolution," generates dynamic selection pressure on POI cycling at the timescale of tens of minutes. We used it to evolve 19 new variants of the LOV transcription factor El222, including in vivo green-light-responsive variants allowing LOV color-multiplexing. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing phycocyanobilin (PCB) unnecessary. Finally, we demonstrated the generality of the method by evolving a non-light-responsive AND gate (PEST-rtTA). Optovolution makes difficult-to-engineer protein functionalities continuously evolvable.
5.
Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells.
Abstract:
Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways to deliver light into cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a genetically encoded optogenetic circuit for the control of gene expression. Furthermore, we utilized a magneto-sensitive protein to engineer a split-protein version of this luminescent enzyme, where its reconstitution is driven by a 50 mT magnetic stimulus. Thus, resulting in a gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.
6.
ShineGAL4 drivers for tissue and cell-type specific optogenetics in Drosophila.
Abstract:
An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.
7.
Single-cell characterization of bacterial optogenetic Cre recombinases.
Abstract:
Microbial optogenetic tools can regulate gene expression with spatial and temporal precision, offering excellent potential for single-cell resolution studies. However, bacterial optogenetic systems have primarily been deployed for population-level experiments. It is not always clear how these tools perform in single cells, where stochastic effects can be substantial. In this study, we focus on optogenetic Cre recombinase and compare the performance of three variants (OptoCre-REDMAP, OptoCre-Vvd, and PA-Cre) for their population-level and single-cell activity. We quantify recombination efficiency, expression variability, and activation dynamics using reporters which produce changes in fluorescence or antibiotic resistance following light-induced Cre activity. We find that optogenetic recombinase performance can be reporter-dependent. Further, single-cell analysis reveals highly heterogeneous activity, with substantial variation in the efficiency and timing of recombinase activity from cell to cell. These findings suggest important criteria for selecting optogenetic recombinases and indicate areas for optimization to improve single-cell capabilities of bacterial optogenetic tools.
8.
Optogenetic manipulation of estrogen receptor signaling to improve estrogen deficiency.
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Liu, J
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Xie, L
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Wang, J
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Chen, Q
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Zhu, M
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Zhang, L
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Xie, S
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Lu, B
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Chen, X
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Xu, Y
Abstract:
Estrogen receptor (ER)-mediated genomic actions are crucial for maintaining various physiological functions, and their dysfunction is associated with numerous human diseases. Traditional estrogen replacement therapy (ERT) is commonly used to manage estrogen deficiency-related conditions, such as vulvovaginal atrophy during menopause, but its systemic effects pose notable risks. This study introduces OptoER, an optogenetic tool engineered to precisely modulate ER-mediated genomic pathways through light-induced transcription regulation, offering spatial-temporal control over ER-dependent gene expression. Our in vitro studies demonstrate that OptoER significantly enhances ER-specific gene transcription and protein synthesis, leading to improved cell proliferation and migration. In a proof-of-principle study using ovariectomized (OVX) mice, OptoER demonstrated considerable therapeutic potential for vaginal atrophy, with observed improvement in epithelial thickness and keratinization. These findings suggest that OptoER provides a targeted therapeutic strategy for estrogen deficiency conditions, with significant implications for treating vaginal atrophy and promoting regenerative healing in estrogen-deprived tissues.
9.
Engineering a High-Activity Photosensitive Synthase for Optogenetic Control of c-di-GMP and Biofilm Dynamics.
Abstract:
Bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a crucial role in bacterial signaling pathways, allowing bacterial cells to respond to various environmental stimuli. The prevalence of c-di-GMP and its potential applications underscore the necessity for developing tools and methods to regulate intracellular c-di-GMP levels. Optogenetic control of c-di-GMP dynamics is particularly attractive because it enables tunable and spatiotemporal regulation of c-di-GMP metabolism. The development of sensitive optogenetic control systems requires highly active, light-responsive c-di-GMP synthases. Here, we report an engineered, highly active photosensitive c-di-GMP synthase, BphS-13. This engineered c-di-GMP synthase was developed from a near-infrared (NIR) light-activable bacteriophytochrome c-di-GMP synthase, BphS, using a three-step directed evolution process that included error-prone PCR, in vitro homologous recombination, and site-directed mutagenesis. After two rounds of this directed evolution strategy, we generated a BphS variant with 13 mutations, referred to as BphS-13. The diguanylate cyclase (DGC) activity of BphS-13 was approximately 13 times higher than that of the original BphS, and it exhibited tightly regulated DGC activity in response to NIR light with minimal leakage in the dark. We then demonstrated the effectiveness of BphS-13 in controlling biofilm dynamics. Overall, this study highlights BphS-13 as a highly active and photosensitive tool for optogenetic applications in biotechnology and suggests its future potential application in mammalian systems for precise control of gene expression, particularly given the lack of native c-di-GMP signaling pathways in mammalian cells.
10.
A dCas9-integrated iLight9O system enables dynamic regulation for enhanced patchoulol biosynthesis in Saccharomyces cerevisiae.
Abstract:
Numerous organisms have evolved the ability to utilize light through photoreceptor proteins that mediate diverse biological processes. Currently, several optogenetic sensor systems are widely used in yeast. However, when these systems are applied for gene repression to regulate endogenous yeast gene expression, they typically require the insertion of corresponding target sites near the native promoter of the gene of interest to achieve precise modulation. To address these constraints, a novel blue light-inducible optogenetic tool designated iLight9 was developed, a single-component optogenetic biosensor integrated with the CRISPR-dCas9 platform. The stability of the iLight9 system was further enhanced by employing a strategy involving the addition of a protein degradation tag. The resulting system was designated as iLight9O, which facilitated programmable regulation of distinct genes through the introduction of specific sgRNAs. Subsequently, systematic metabolic engineering strategies were employed to construct an efficient patchoulol-producing cell factory in Saccharomyces cerevisiae. Moreover, a two-step isoprenol utilization (IU) pathway was introduced into the recombinant strain to enhance its capacity for patchoulol biosynthesis. Crucially, the iLight9O system was adopted to dynamically downregulate squalene synthase, a key enzyme in the competing squalene biosynthetic pathway. This optogenetic flux control strategy increased patchoulol titers by 66 % in the IU-optimized strain and 24 % in the MVAIU2 strain, demonstrating significant improvements over static engineering approaches.
11.
Rapid optogenetic manipulation of autophagy reveals that the nuclear pore complex is a robust autophagy substrate.
Abstract:
Autophagy, a conserved recycling process, manages intracellular quality control to mitigate stress. To determine the rapid effects of autophagy perturbation, we developed the first optogenetic tool to rapidly inhibit autophagy, termed ASAP. Our approach selectively inhibits autophagy within 5 minutes, providing a precise and dynamic approach to study autophagy regulation. Proteomic profiling with ASAP revealed the most tightly regulated autophagy substrates along with novel, previously unidentified substrates, including nuclear pore complex (NPC) proteins. Interestingly, autophagy regulates quality control of incomplete NPCs still in the cytoplasm via specific LC3-interacting regions (LIRs), sparing NPCs embedded in the nuclear envelope. Upon rapid autophagy inhibition, incomplete NPCs accumulate and instead of undergoing autophagic degradation, cytoplasmic NPCs aggregate in processing bodies. Using ASAP, we demonstrate rapid and specific inhibition of autophagy, revealing that the nuclear pore complex is a tightly regulated autophagy substrate.
12.
On-demand cancer immunotherapy via single-cell encapsulation of synthetic circuit-engineered cells.
Abstract:
Despite the therapeutic potential of engineered immune cell therapy against metastases, it faces challenges including cytokine-driven systemic toxicity, off-target biodistribution, and host rejection. Here, we develop red/far-red light-regulated individually encapsulated (RL/FRL-EnE) cells, integrating optogenetics with biomaterial encapsulation for precise immunomodulation. This system uses a phytochrome A-based photoswitch (ΔPhyA-PCB) that enables bidirectional control. RL (660 nanometers) triggers interferon-γ, interleukin-6, and anti-CD47 expression via ΔPhyA-PCB-far-red elongated hypocotyl 1 heterodimerization, while FRL (740 nanometers) rapidly reverses production, minimizing toxicity. Single-cell nanoencapsulation prevents intercellular cross-talk and immune clearance, enabling strict light-dependent regulation and extended tumor residence. In vivo, RL/FRL-EnE cells remodeled the tumor microenvironment, reducing immunosuppressive myeloid cells (1.3- to 1.7-fold), while enhancing dendritic cell (1.4-fold) and CD8+ T cell (2.8-fold) infiltration. Collectively, this work establishes a paradigm for closed-loop cellular immunotherapy, where light-regulated living therapeutics achieve on-demand immune reprogramming.
13.
Optogenetic Proximity Labeling Maps Spatially Resolved Mitochondrial Surface Proteomes and a Locally Regulated Ribosome Pool.
Abstract:
Outer mitochondrial membranes (OMM) function as dynamic hubs for inter-organelle communication, integrating bidirectional signals, and coordinating organelle behavior in a context-dependent manner. However, tools for mapping mitochondrial surface proteomes with high spatial and temporal resolution remain limited. Here, we introduce an optogenetic proximity labeling strategy using LOV-Turbo, a light-activated biotin ligase, to profile mitochondrial surface proteomes with improved precision, temporal control, and reduced background. By fusing LOV-Turbo to a panel of variants of an OMM-anchored protein, Miro1, we generate spatially distinct baits that resolve modular architectures and regulatory states of the OMM proteomes across diverse conditions, a database we name MitoSurf. Building on this proteomic map, we present RiboLOOM, a platform that defines LOV-Turbo labeled ribosomes and their bound mRNAs at the mitochondrial surface. MitoSurf and RiboLOOM uncover a spatially distinct ribosome pool at the OMM that is maintained by Miro1, enabling local mRNA engagement and translation of mitochondria-related proteins. These findings establish Miro1 as a key organizer of mitochondrial protein biogenesis through spatial confinement of surface-associated ribosomes. Our platform reveals an uncharted layer of mitochondrial surface biology and provides a generalizable strategy to dissect dynamic RNA-protein-organelle interfaces in living cells.
14.
Single copy optogenetic system for Streptomyces.
Abstract:
LitR is a blue-green light-sensing transcriptional regulator that uses coenzyme B12 as a chromophore. In this study, we developed a genome-integrative light-inducible expression (iLiEX) system in Streptomyces griseus NBRC 13350, a Gram-positive bacterium that produces streptomycin. The system incorporates LitR, transcriptional amplification module T7 RNA polymerase, and a serine integrase. Using iLiEX, we achieved light-dependent overproduction of catechol-2,3-dioxygenase and β-glucuronidase (GUS) at levels comparable to those from a high-copy plasmid. Notably, GUS activity was 39-fold higher than with the constitutively strong ermE* promoter. The iLiEX system was also functional in S. coelicolor, S. lividans, S. albus J1074, and S. avermitilis. We improved iLiEX in two key ways: by optimizing the ribosome-binding site of T7 RNA polymerase to increase expression, and by introducing the T7 lysozyme gene to reduce leaky transcription. The system's versatility was improved by shortening the T7 promoter from 89 to 44 bp. For simple visualization on agar plates, light-dependent overexpression of fluorescent proteins, a chromogenic protein, and a brown pigment synthesis enzyme was demonstrated. High-level production of secreted enzymes, including laccase and transglutaminase, was also confirmed. Overall, we developed a single-copy light-inducible overexpression system with broad functionality across multiple Streptomyces species.
15.
Optogenetic Control the Activity of Pyruvate Decarboxylase in Saccharomyces cerevisiae for Tunable Ethanol Production.
Abstract:
Saccharomyces cerevisiae is a widely used chassis in metabolic engineering. Due to the Crabtree effect, it preferentially produces ethanol under high-glucose conditions, limiting the synthesis of other valuable metabolites. Conventional metabolic engineering approaches typically rely on irreversible genetic modifications, making it insufficient for dynamic metabolic control. In contrast, optogenetics offers a reversible and tunable method for regulating cellular metabolism with high temporal precision. In this study, we engineered the pyruvate decarboxylase isozyme 1 (Pdc1) by inserting the photosensory modules (AsLOV2 and cpLOV2 domains) into rationally selected positions within the enzyme. Through a growth phenotype-based screening system, we identified two blue light-responsive variants, OptoPdc1D1 and OptoPdc1D2, which enable light-dependent control of enzymatic activity. Leveraging these OptoPdc1 variants, we developed opto-S. cerevisiae strains, MLy-9 and MLy-10, which demonstrated high efficiency in modulating both cell growth and ethanol production. These strains allow reliable regulation of ethanol biosynthesis in response to blue light, achieving a dynamic control range of approximately 20- to 120-fold. The opto-S. cerevisiae strains exhibited dose-dependent production in response to blue light intensity and pulse patterns, confirming their potential for precise metabolic control. This work establishes a novel protein-level strategy for regulating metabolic pathways in S. cerevisiae and introduces an effective method for controlling ethanol metabolism via optogenetic regulation.
16.
Improving T cell expansion by optogenetically engineered bacteria-loaded MMP-2-responsive cyclophosphamide for antitumor immunotherapy.
Abstract:
The efficacy of antitumor immunotherapy is closely associated with the expansion of tumor-infiltrating CD8+ T cells. However, within the tumor microenvironment, CD8+ T cells often exhibit reduced proliferation due to persistent exposure to tumor antigens. The cytokine IL-2 is a potent growth factor that can drive the expansion of tumor-infiltrating lymphocytes. While its clinical application has been severely limited by systemic toxicity and in vivo instability. To address these challenges, we have developed a dual-responsive system (EcNIL-2@UCNP/Gel-CTX) leveraging the hypoxic tropisms of E. coli Nissle 1917(EcN). This system is capable of producing IL-2 in situ upon near-infrared (NIR) irradiation and releasing low-dose cyclophosphamide (CTX) in response to matrix metalloproteinase-2 (MMP-2) in the tumor microenvironment. The EcNIL-2@UCNP/Gel-CTX system not only drives the expansion of CD8+ T cells and boost the activity of NK cells but also reduces Treg cell populations, thereby remodeling the immune microenvironment and eliciting robust tumor-specific immune responses in H22 subcutaneous tumors in mice and confers long-term protection against tumor rechallenge by promoting the generation of durable memory T cells. Our findings provide an both light and tumor microenvironment responsive platform for enhanced cancer immunotherapy.
17.
Phase-driven rewiring in Escherichia coli enhances coenzyme Q10 biosynthesis via temporal and energetic coordination.
Abstract:
Coenzyme Q10 biosynthesis in Escherichia coli is constrained by kinetic mismatches between precursor synthesis and methylation, alongside bioenergetic uncoupling. We implemented an optogenetic phase-control strategy integrating dynamic light induction, ribosome binding site (RBS) engineering, and real-time membrane potential (ΔΨ) feedback. Temporal coordination of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and UbiG methyltransferase (UbiG) via a 6-h phase delay reduced methylglyoxal shunt flux by 41 ± 3% (p < 0.01) through enhanced precursor channeling. Membrane hyperpolarization to - 90 ± 2 mV (relative to - 70 mV in controls) triggered voltage-gated UbiG membrane localization (62 ± 3%) and ATP-driven S-adenosylmethionine regeneration, increasing methylation efficiency 2.3-fold. Multivariate modeling identified ΔΨ and acetate as critical control parameters, enabling optimized fermentation (dissolved oxygen (DO) 15-20%, pH 6.7-6.9). The engineered strain achieved 0.63 ± 0.07 g/L CoQ10 in 5-L bioreactors-a 4.3-fold improvement over the static control strain (0.15 ± 0.02 g/L)-with 82.5% carbon efficiency and 25.8% glycerol-to-product yield. This work establishes bioenergetically coupled temporal control as a scalable paradigm for membrane-bound isoprenoid biomanufacturing. KEY POINTS: • Phase-driven enzyme synchronization via optogenetics resolves kinetic mismatch. • Membrane hyperpolarization gates enzyme localization and ATP regeneration. • Model-integrated bioenergetic-process control enhances CoQ10 production efficiency.
18.
Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells.
Abstract:
Precise control of gene expression is one of the fundamental goals of synthetic biology. Whether the objective is to modify endogenous cellular function or induce the expression of molecules for diagnostic and therapeutic purposes, gene regulation remains a key aspect of biological systems. Over time, advances in protein engineering and molecular biology have led to the creation of gene circuits capable of inducing the expression of specific proteins in response to external stimulus such as light. These optogenetic, or light-activated circuits hold significant potential for gene therapy as a tool for regulating the expression of therapeutic genes within cells. However, the applications of optogenetic systems can be limited by the lack of efficient ways for light delivery inside cells or tissue. Our approach to address this challenge is to harness the power of bioluminescence to produce light directly inside cells using a luminescent enzyme. Combined with a photosensitive transcription factor, we report the development of a fully genetically encoded optogenetic circuit for control of gene expression. Furthermore, we utilized a magneto sensitive protein to engineer a split protein version of this luminescent enzyme, where its reconstitution is driven by a 50mT magnetic stimulus. Thus, resulting in a first-of-its-kind gene circuit activated by a combination of light and magnetic stimulus. We expect this work to advance the implementation of light-controlled systems without the need of external light sources, as well as serve as a basis for the development of future magneto-sensitive tools.
19.
An Engineered Living Material with pro-angiogenic activity inducible by near-infrared light.
Abstract:
Impaired angiogenesis is a central barrier in the treatment of chronic and deep tissue wounds, preventing progression through the normal healing cascade. While the combination of near-infrared (NIR) photobiomodulation and pro-angiogenic growth factors has shown synergistic therapeutic benefit, the clinical translation of growth factor therapy is hindered by high cost, instability and the need for localized dosing to avoid aberrant vasculature. Peptidomimetics such as the VEGF-derived QK peptide offer a more stable and predictable alternative, but still require a means for localized, tunable presentation. Here, we establish an engineered living material based delivery system that responds to clinically relevant NIR light to produce and releases a QK-Fusion protein directly at the target site. The probiotic Escherichia coli Nissle 1917 was engineered with an 800 nm-responsive optogenetic circuit and encapsulated within an optimized alginate core–shell hydrogel that ensures biocontainment while allowing controlled outward diffusion of the secreted peptide. The released peptide remains non-cytotoxic and capable of binding extracellular matrix analogs and promoting the formation of organized, branched capillary-like networks in endothelial cultures. We thus establish a strategy for developing engineered living materials towards remote-controlled angiogenic stimulation.
20.
A tool for modeling gene regulatory networks (GRN_modeler) and its applications to synthetic biology.
Abstract:
Modeling and simulating gene regulatory networks (GRNs) is crucial for understanding biological processes, predicting system behavior, interpreting experimental data and guiding the design of synthetic systems. In synthetic biology, GRNs are fundamental to enable the design and control of complex functions. However, GRN simulations can be time-consuming and often require specialized expertise. To address this challenge, we developed GRN_modeler - a user-friendly tool with a graphical user interface that enables users without programming experience to create phenomenological models, while also offering command-line support for advanced users. GRN_modeler supports the analysis of both dynamical behaviors and spatial pattern formation. We demonstrate its versatility through several examples in synthetic biology, including the design of novel oscillator families capable of robust oscillation with an even number of nodes, complementing the classical repressilator family, which requires odd-numbered nodes. Furthermore, we showcase how GRN_modeler allowed us to develop a light-detecting biosensor in Escherichia coli that tracks light intensity over several days and leaves a record in the form of ring patterns in bacterial colonies.
21.
Photoswitchable intein for light control of covalent protein binding and cleavage.
Abstract:
Precise control of covalent protein binding and cleavage in mammalian cells is crucial for manipulating cellular processes but remains challenging due to dark background, poor stability, low efficiency, or requirement of unnatural amino acids in current optogenetic tools. We introduce a photoswitchable intein (PS Intein) engineered by allosterically modulating a small autocatalytic gp41-1 intein with tandem Vivid photoreceptor. PS Intein exhibits superior functionality and low background in cells compared to existing tools. PS Intein-based systems enable light-induced covalent binding, cleavage, and release of proteins for regulating gene expression and cell fate. The high responsiveness and ability to integrate multiple inputs allow for intersectional cell targeting using cancer- and tumor microenvironment-specific promoters. PS Intein tolerates various fusions and insertions, facilitating its application in diverse cellular contexts. This versatile technology offers efficient light-controlled protein manipulation, providing a powerful tool for adding functionalities to proteins and precisely controlling protein networks in living cells.
22.
Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation.
Abstract:
Localized translation broadly enables spatiotemporal control of gene expression. Here, we present LOV-domain-controlled ligase for translation localization (LOCL-TL), an optogenetic approach for monitoring translation with codon resolution at any defined subcellular location under physiological conditions. Application of LOCL-TL to mitochondrially localized translation revealed that ∼20% of human nuclear-encoded mitochondrial genes are translated on the outer mitochondrial membrane (OMM). Mitochondrially translated messages form two classes distinguished by encoded protein length, recruitment mechanism, and cellular function. An evolutionarily ancient mechanism allows nascent chains to drive cotranslational recruitment of long proteins via an unanticipated bipartite targeting signal. Conversely, mRNAs of short proteins, especially eukaryotic-origin electron transport chain (ETC) components, are specifically recruited by the OMM protein A-kinase anchoring protein 1 (AKAP1) in a translation-independent manner that depends on mRNA splicing. AKAP1 loss lowers ETC levels. LOCL-TL thus reveals a hierarchical strategy that enables preferential translation of a subset of proteins on the OMM.
23.
De novo designed protein guiding targeted protein degradation.
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Li, Z
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Qiao, G
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Wang, X
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Wang, M
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Cheng, J
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Hu, G
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Li, X
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Wu, J
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Liu, J
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Gao, C
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Liu, L
Abstract:
Targeted protein degradation is a powerful tool for biological research, cell therapy, and synthetic biology. However, conventional methods often depend on pre-fused degrons or chemical degraders, limiting their wider applications. Here we develop a guided protein labeling and degradation system (GPlad) in Escherichia coli, using de novo designed guide proteins and arginine kinase (McsB) for precise degradation of various proteins, including fluorescent proteins, metabolic enzymes, and human proteins. We expand GPlad into versatile tools such as antiGPlad, OptoGPlad, and GPTAC, enabling reversible inhibition, optogenetic regulation, and biological chimerization. The combination of GPlad and antiGPlad allows for programmable circuit construction, including ON/OFF switches, signal amplifiers, and oscillators. OptoGPlad-mediated degradation of MutH accelerates E. coli evolution under protocatechuic acid stress, reducing the required generations from 220 to 100. GPTAC-mediated degradation of AroE enhanced the titer of 3-dehydroshikimic acid to 92.6 g/L, a 23.8% improvement over the conventional CRISPR interference method. We provide a tunable, plug-and-play strategy for straightforward protein degradation without the need for pre-fusion, with substantial implications for synthetic biology and metabolic engineering.
24.
Deep-tissue high-sensitivity multimodal imaging and optogenetic manipulation enabled by biliverdin reductase knockout.
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Kasatkina, LA
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Ma, C
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Sheng, H
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Lowerison, M
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Menozzi, L
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Baloban, M
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Tang, Y
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Xu, Y
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Humayun, L
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Vu, T
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Song, P
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Yao, J
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Verkhusha, VV
Abstract:
Performance of near-infrared probes and optogenetic tools derived from bacterial phytochromes is limited by availability of their biliverdin chromophore. To address this, we use a biliverdin reductase-A knock-out mouse model (Blvra-/-), which elevates endogenous biliverdin levels. We show that Blvra⁻/⁻ significantly enhances function of bacterial phytochrome-based systems. Light-controlled transcription using iLight optogenetic tool improves ~25-fold in Blvra-/- cells, compared to wild-type controls, and achieves ~100-fold activation in neurons. Light-induced insulin production in Blvra-/- mice reduces blood glucose by ~60% in diabetes model. To overcome depth limitations in imaging, we employ 3D photoacoustic, ultrasound, and two-photon fluorescence microscopy. This enables simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of brain vasculature at depths of ~7 mm through intact scalp and skull. Two-photon microscopy achieves cellular resolution of miRFP720-expressing neurons at ~2.2 mm depth. Overall, Blvra-/- model represents powerful platform for improving efficacy of biliverdin-dependent tools for deep-tissue imaging and optogenetic manipulation.
25.
Optogenetic-Controlled iPSC-Based Vaccines for Prophylactic and Therapeutic Tumor Suppression in Mice.
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Qiao, L
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Niu, L
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Wang, Z
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Dai, D
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Tang, S
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Ma, X
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Deng, Z
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Yu, G
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Zhou, Y
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Yan, T
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Liu, X
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Kong, D
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Hu, L
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Li, X
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Zhao, J
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Cai, F
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Wang, M
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Ye, H
Abstract:
Induced pluripotent stem cells (iPSCs) share similar cellular features and various antigens profiles with cancer cells. Leveraging these characteristics, iPSCs hold great promise for developing wide-spectrum vaccines against cancers. In practice, iPSCs are typically combined with immune adjuvants to enhance antitumor immune responses; however, traditional adjuvants lack controllability and can induce systemic toxicity, which has limited their broad application. Here, a red/far-red light-controlled iPSC-based vaccine (RIVA) based on the chimeric photosensory protein FnBphP and its interaction partner LDB3 is developed; RIVA preserves the intrinsic tumor antigens of iPSCs and enables optogenetic control of an immune adjuvant's (IFN-β) expression under red light illumination. Experiments in multiple mouse tumor models demonstrate that RIVA inhibits tumor growth and improves animal survival in prophylactic and therapeutic settings, including against pulmonary metastatic 4T1 breast cancer. RIVA efficiently stimulates dendritic cell maturation, eliciting innate immune activation effects through NK cells and elicit adaptive immune anti-tumor responses through CD4+ and CD8+ T cells. Moreover, RIVA protects animals against tumor re-challenge by inducing strong immunological memory, with minimal systemic toxicity. This study demonstrates RIVA as an effective optogenetic approach for developing safe multi-antigen vaccines for the prevention and treatment of cancer.
