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 111 results
1.

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

blue PAL E. coli Transgene expression
bioRxiv, 31 Jul 2022 DOI: 10.1101/2022.07.30.502174 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 ten-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that generally 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.
2.

Dynamic cybergenetic control of bacterial co-culture composition via optogenetic feedback.

blue Magnets E. coli Transgene expression
bioRxiv, 14 Jun 2022 DOI: 10.1101/2022.06.13.495893 Link to full text
Abstract: Communities of microbes play important roles in natural environments and hold great potential for deploying division-of-labor strategies in synthetic biology and bioproduction. However, the difficulty of controlling the composition of microbial consortia over time hinders their optimal use in many applications. Here, we present a fully automated, high-throughput platform that combines real-time measurements and computer-controlled optogenetic modulation of bacterial growth to implement precise and robust compositional control of a two-strain E. coli community. Additionally, we develop a general framework for dynamic modeling of synthetic genetic circuits in the physiological context of E. coli and use a host-aware model to determine the optimal control parameters of our closed-loop compositional control system. Our platform succeeds in stabilizing the strain ratio of multiple parallel co-cultures at arbitrary levels and in changing these targets over time, opening the door for the implementation of dynamic compositional programs in synthetic bacterial communities.
3.

A red light-responsive photoswitch for deep tissue optogenetics.

near-infrared red BphP1/Q-PAS1 DrBphP MagRed HEK293T HeLa in vitro Neuro-2a Transgene expression
Nat Biotechnol, 13 Jun 2022 DOI: 10.1038/s41587-022-01351-w Link to full text
Abstract: Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light-activatable photoswitch that consists of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light-activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light-inducible transcriptional regulators based on CRISPR-Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.
4.

A Single-Component Blue Light-Induced System Based on EL222 in Yarrowia lipolytica.

blue EL222 Y. lipolytica Transgene expression
Int J Mol Sci, 6 Jun 2022 DOI: 10.3390/ijms23116344 Link to full text
Abstract: Optogenetics has the advantages of a fast response time, reversibility, and high spatial and temporal resolution, which make it desirable in the metabolic engineering of chassis cells. In this study, a light-induced expression system of Yarrowia lipolytica was constructed, which successfully achieved the synthesis and functional verification of Bleomycin resistance protein (BleoR). The core of the blue light-induced system, the light-responsive element (TF), is constructed based on the blue photosensitive protein EL222 and the transcription activator VP16. The results show that the light-induced sensor based on TF, upstream activation sequence (C120)5, and minimal promoter CYC102 can respond to blue light and initiate the expression of GFPMut3 report gene. With four copies of the responsive promoter and reporter gene assembled, they can produce a 128.5-fold higher fluorescent signal than that under dark conditions after 8 h of induction. The effects of light dose and periodicity on this system were investigated, which proved that the system has good spatial and temporal controllability. On this basis, the light-controlled system was used for the synthesis of BleoR to realize the expression and verification of functional protein. These results demonstrated that this system has the potential for the transcriptional regulation of target genes, construction of large-scale synthetic networks, and overproduction of the desired product.
5.

Optogenetic Protein Cleavage in Zebrafish Embryos.

violet PhoCl HEK293T HeLa zebrafish in vivo Transgene expression
bioRxiv, 24 May 2022 DOI: 10.1101/2022.05.23.493165 Link to full text
Abstract: A wide array of optogenetic tools is available that allow for precise spatiotemporal control over many cellular processes. These tools have been especially popular among zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and apoptosis.
6.

Light-induced fermenter production of derivatives of the sweet protein monellin is maximized in prestationary Saccharomyces cerevisiae cultures.

blue CRY2/CIB1 S. cerevisiae Transgene expression
Biotechnol J, 28 Apr 2022 DOI: 10.1002/biot.202100676 Link to full text
Abstract: Optogenetics has great potential for biotechnology and metabolic engineering due to the cost-effective control of cellular activities. The usage of optogenetics techniques for the biosynthesis of bioactive molecules ensures reduced costs and enhanced regulatory possibilities. This requires development of efficient methods for light-delivery during a production process in a fermenter. Here, we benchmarked the fermenter production of a low-caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. An expression system based on the light-controlled interaction between Cry2 and Cib1 was used for sweet-protein production. Optimization of the fermenter process was achieved by increasing the light-flux during the production phase to circumvent shading by yeast cells at high densities. Maximal amounts of the sweet-protein were produced in a pre-stationary growth phase, whereas at later stages, a decay in protein abundance was observable. Our investigation showcases the upscaling of an optogenetic production process from small flasks to a bioreactor. Optogenetic-controlled production in a fermenter is highly cost-effective due to the cheap inducer and therefore a viable alternative to chemicals for a process that requires an induction step.
7.

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.
8.

CRY-BARs: Versatile light-gated molecular tools for the remodeling of membrane architectures.

blue CRY2/CRY2 HEK293T primary mouse cortical neurons Control of cytoskeleton / cell motility / cell shape Transgene expression Neuronal activity control
bioRxiv, 17 Mar 2022 DOI: 10.1101/2022.01.28.478241 Link to full text
Abstract: BAR (Bin, Amphiphysin and Rvs) protein domains are responsible for the generation of membrane curvature and represent a critical mechanical component of cellular functions. Thus, BAR domains have great potential as components of membrane-remodeling tools for cell biologists. In this work, we describe the design and implementation of a family of versatile light-gated I-BAR domain containing tools (CRY-BARs) with applications in the remodeling of membrane architectures and the control of cellular dynamics. By taking advantage of the intrinsic membrane binding propensity of the I-BAR domain, CRY-BARs can be used for spatial and temporal control of cellular processes that require induction of membrane protrusions. Using cell lines and primary neuron cultures, we demonstrate that the CRY-BAR optogenetic tool reports membrane dynamic changes associated with cellular activity. Moreover, we provide evidence that Ezrin acts as a relay between the plasma membrane and the actin cytoskeleton and therefore is an important mediator of switch function. Overall, CRY-BARs hold promise as a useful addition to the optogenetic toolkit to study membrane remodeling in live cells.
9.

Using single-cell models to predict the functionality of synthetic circuits at the population scale.

blue EL222 S. cerevisiae Transgene expression
Proc Natl Acad Sci U S A, 10 Mar 2022 DOI: 10.1073/pnas.2114438119 Link to full text
Abstract: SignificanceAt the single-cell level, biochemical processes are inherently stochastic. For many natural systems, the resulting cell-to-cell variability is exploited by microbial populations. In synthetic biology, however, the interplay of cell-to-cell variability and population processes such as selection or growth often leads to circuits not functioning as predicted by simple models. Here we show how multiscale stochastic kinetic models that simultaneously track single-cell and population processes can be obtained based on an augmentation of the chemical master equation. These models enable us to quantitatively predict complex population dynamics of a yeast optogenetic differentiation system from a specification of the circuit's components and to demonstrate how cell-to-cell variability can be exploited to purposefully create unintuitive circuit functionality.
10.

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.
11.

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.
12.

Bifunctional optogenetic switch for improving shikimic acid production in E. coli.

blue VVD E. coli Transgene expression
Biotechnol Biofuels, 7 Feb 2022 DOI: 10.1186/s13068-022-02111-3 Link to full text
Abstract: Background Biomass formation and product synthesis decoupling have been proven to be promising to increase the titer of desired value add products. Optogenetics provides a potential strategy to develop light-induced circuits that conditionally control metabolic flux redistribution for enhanced microbial production. However, the limited number of light-sensitive proteins available to date hinders the progress of light-controlled tools. Results To address these issues, two optogenetic systems (TPRS and TPAS) were constructed by reprogramming the widely used repressor TetR and protease TEVp to expand the current optogenetic toolkit. By merging the two systems, a bifunctional optogenetic switch was constructed to enable orthogonally regulated gene transcription and protein accumulation. Application of this bifunctional switch to decouple biomass formation and shikimic acid biosynthesis allowed 35 g/L of shikimic acid production in a minimal medium from glucose, representing the highest titer reported to date by E. coli without the addition of any chemical inducers and expensive aromatic amino acids. This titer was further boosted to 76 g/L when using rich medium fermentation. Conclusion The cost effective and light-controlled switch reported here provides important insights into environmentally friendly tools for metabolic pathway regulation and should be applicable to the production of other value-add chemicals.
13.

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.
14.

Development of Optogenetic Dual-Switch System for Rewiring Metabolic Flux for Polyhydroxybutyrate Production.

blue green CcaS/CcaR EL222 RsLOV YtvA E. coli Transgene expression
Molecules, 18 Jan 2022 DOI: 10.3390/molecules27030617 Link to full text
Abstract: Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10-CcaS#10-CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10-CcaS#10-CcaR system was combined with a blue light-activated YF1-FixJ-PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.
15.

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.
16.

Regulating bacterial behavior within hydrogels of tunable viscoelasticity.

blue YtvA E. coli Transgene expression
bioRxiv, 6 Jan 2022 DOI: 10.1101/2022.01.06.475183 Link to full text
Abstract: Engineered living materials (ELMs) are a new class of materials in which living organism incorporated into diffusive matrices uptake a fundamental role in material’s composition and function. Understanding how the spatial confinement in 3D affects the behavior of the embedded cells is crucial to design and predict ELM’s function, regulate and minimize their environmental impact and facilitate their translation into applied materials. This study investigates the growth and metabolic activity of bacteria within an associative hydrogel network (Pluronic-based) with mechanical properties that can be tuned by introducing a variable degree of acrylate crosslinks. Individual bacteria distributed in the hydrogel matrix at low density form functional colonies whose size is controlled by the extent of permanent crosslinks. With increasing stiffness and decreasing plasticity of the matrix, a decrease in colony volumes and an increase in their sphericity is observed. Protein production surprisingly follows a different pattern with higher production yields occurring in networks with intermediate permanent crosslinking degrees. These results demonstrate that, bacterial mechanosensitivity can be used to control and regulate the composition and function of ELMs by thoughtful design of the encapsulating matrix, and by following design criteria with interesting similarities to those developed for 3D culture of mammalian cells.
17.

Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins.

blue CRY2/CIB1 PAL VVD HEK293T HeLa Transgene expression Epigenetic modification Endogenous gene expression
Nat Biotechnol, 3 Jan 2022 DOI: 10.1038/s41587-021-01112-1 Link to full text
Abstract: RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR-Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.
18.

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.
19.

Optogenetic operated probiotics to regulate host metabolism by mimicking enteroendocrine.

blue YtvA L. lactis Transgene expression
bioRxiv, 1 Dec 2021 DOI: 10.1101/2021.11.30.470589 Link to full text
Abstract: The enteroendocrine system plays an important role in metabolism. The gut microbiome regulates enteroendocrine in an extensive way, arousing attention in biomedicine. However, conventional strategies of enteroendocrine regulation via gut microbiome are usually non-specific or imprecise. Here, an optogenetic operated probiotics system was developed combining synthetic biology and flexible electronics to achieve in situ controllable secretion to mimic enteroendocrine. Firstly, optogenetic engineered Lactococcus lactis (L. lactis) were administrated in the intestinal tract. A wearable optogenetic device was designed to control optical signals remotely. Then, L. lactis could secrete enteroendocrine hormone according to optical signals. As an example, optogenetic L. lactis could secrete glucagon-like peptide-1(GLP-1) under the control of the wearable optogenetic device. To improve the half-life of GLP-1 in vivo, the Fc domain from immunoglobulin was fused. Treated with this strategy, blood glucose, weight and other features were relatively well controlled in rats and mice models. Furthermore, up-conversion microcapsules were introduced to increase the excitation wavelength of the optogenetic system for better penetrability. This strategy has biomedical potential in metabolic diseases therapy by mimicking enteroendocrine.
20.

Rapid and robust optogenetic control of gene expression in Drosophila.

blue Magnets D. melanogaster in vivo Transgene expression Endogenous gene expression
Dev Cell, 29 Nov 2021 DOI: 10.1016/j.devcel.2021.11.016 Link to full text
Abstract: Deciphering gene function requires the ability to control gene expression in space and time. Binary systems such as the Gal4/UAS provide a powerful means to modulate gene expression and to induce loss or gain of function. This is best exemplified in Drosophila, where the Gal4/UAS system has been critical to discover conserved mechanisms in development, physiology, neurobiology, and metabolism, to cite a few. Here we describe a transgenic light-inducible Gal4/UAS system (ShineGal4/UAS) based on Magnet photoswitches. We show that it allows efficient, rapid, and robust activation of UAS-driven transgenes in different tissues and at various developmental stages in Drosophila. Furthermore, we illustrate how ShineGal4 enables the generation of gain and loss-of-function phenotypes at animal, organ, and cellular levels. Thanks to the large repertoire of UAS-driven transgenes, ShineGal4 enriches the Drosophila genetic toolkit by allowing in vivo control of gene expression with high temporal and spatial resolutions.
21.

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.
22.

Implementation of a novel optogenetic tool in mammalian cells based on a split T7 RNA polymerase.

blue Magnets VVD HEK293T Transgene expression
bioRxiv, 27 Oct 2021 DOI: 10.1101/2021.10.27.466068 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 orthogonally control gene expression in mammalian cells. 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 orthogonal optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool 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 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 gene expression burden when compared to other optogenetic constructs. These properties make mOptoT7 a new powerful tool to use when orthogonality and viral-like RNA species are desired in both synthetic biology and basic science applications.
23.

Spatial and temporal control of expression with light-gated LOV-LexA.

blue AsLOV2 D. melanogaster in vivo Schneider 2 Transgene expression Neuronal activity control
bioRxiv, 19 Oct 2021 DOI: 10.1101/2021.10.19.465021 Link to full text
Abstract: The ability to drive expression of exogenous genes in different tissues and cell types, under control of specific enhancers, has catapulted discovery in biology. While many enhancers drive expression broadly, several genetic tricks have been developed to obtain access to isolated cell types. However, studies of topographically organized neuropiles, such as the optic lobe in fruit flies, have raised the need for a system that can access subsets of cells within a single neuron type, a feat currently dependent on stochastic flip-out methods. To access the same subsets of cells consistently across flies, we developed LOV-LexA, a light-gated expression system based on the bacterial LexA transcription factor and the plant-derived LOV photosensitive domain. Expression of LOV-Lex in larval fat body as well as pupal and adult neurons enables spatial and temporal control of expression of transgenes under LexAop sequences with blue light. The LOV-LexA tool thus provides another layer of intersectional genetics, allowing for light-controlled genetic access to the same subsets of cells within an expression pattern across individual flies.
24.

Requirements for mammalian promoters to decode transcription factor dynamics.

blue AsLOV2 HEK293 Transgene expression
bioRxiv, 17 Oct 2021 DOI: 10.1101/2021.10.12.464037 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 them. Combining optogenetics, deep learning-based image analysis and mathematical modeling, we find that 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. Furthermore, we propose a theoretical mechanism based on phase separation that would allow a promoter to be activated better by pulsatile than sustained TF signals. These results provide an understanding on how TF dynamics are decoded in mammalian cells, which is important to develop optimal strategies to counteract disease conditions, and suggest ways to achieve multiplexing in synthetic pathways.
25.

A light tunable differentiation system for the creation and control of consortia in yeast.

blue EL222 S. cerevisiae Transgene expression Cell differentiation
Nat Commun, 5 Oct 2021 DOI: 10.1038/s41467-021-26129-7 Link to full text
Abstract: Artificial microbial consortia seek to leverage division-of-labour to optimize function and possess immense potential for bioproduction. Co-culturing approaches, the preferred mode of generating a consortium, remain limited in their ability to give rise to stable consortia having finely tuned compositions. Here, we present an artificial differentiation system in budding yeast capable of generating stable microbial consortia with custom functionalities from a single strain at user-defined composition in space and in time based on optogenetically-driven genetic rewiring. Owing to fast, reproducible, and light-tunable dynamics, our system enables dynamic control of consortia composition in continuous cultures for extended periods. We further demonstrate that our system can be extended in a straightforward manner to give rise to consortia with multiple subpopulations. Our artificial differentiation strategy establishes a novel paradigm for the creation of complex microbial consortia that are simple to implement, precisely controllable, and versatile to use.
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