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 40 results
1.

Engineering Improved Photoswitches for the Control of Nucleocytoplasmic Distribution.

blue AsLOV2 HeLa in vitro S. cerevisiae Epigenetic modification
ACS Synth Biol, 15 Nov 2018 DOI: 10.1021/acssynbio.8b00368 Link to full text
Abstract: Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control sub-cellular localization and activity. We previously engineered two optogenetic systems, the Light Activated Nuclear Shuttle (LANS) and the Light-Inducible Nuclear eXporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favourably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic 'tool kit' for the research community.
2.

Programming the Dynamic Control of Bacterial Gene Expression with a Chimeric Ligand- and Light-Based Promoter System.

blue EL222 E. coli
ACS Synth Biol, 6 Nov 2018 DOI: 10.1021/acssynbio.8b00280 Link to full text
Abstract: To program cells in a dynamic manner, synthetic biologists require precise control over the threshold levels and timing of gene expression. However, in practice, modulating gene expression is widely carried out using prototypical ligand-inducible promoters, which have limited tunability and spatiotemporal resolution. Here, we built two dual-input hybrid promoters, each retaining the function of the ligand-inducible promoter while being enhanced with a blue-light-switchable tuning knob. Using the new promoters, we show that both ligand and light inputs can be synchronously modulated to achieve desired amplitude or independently regulated to generate desired frequency at a specific amplitude. We exploit the versatile programmability and orthogonality of the two promoters to build the first reprogrammable logic gene circuit capable of reconfiguring into logic OR and N-IMPLY logic on the fly in both space and time without the need to modify the circuit. Overall, we demonstrate concentration- and time-based combinatorial regulation in live bacterial cells with potential applications in biotechnology and synthetic biology.
3.

Discovering selective binders for photoswitchable proteins using phage display.

blue AsLOV2 PYP BEAS-2B
ACS Synth Biol, 11 Sep 2018 DOI: 10.1021/acssynbio.8b00123 Link to full text
Abstract: Nature provides an array of proteins that change conformation in response to light. The discovery of a complementary array of proteins that bind only the light-state or dark-state conformation of their photoactive partner proteins would allow each light-switchable protein to be used as an optogenetic tool to control protein-protein interactions. However, as many photoactive proteins have no known binding partner, the advantages of optogenetic control - precise spatial and temporal resolution - are currently restricted to a few well-defined natural systems. In addition, the affinities and kinetics of native interactions are often sub-optimal and are difficult to engineer in the absence of any structural information. We report a phage display strategy using a small scaffold protein that can be used to discover new binding partners for both light and dark states of a given light-switchable protein. We used our approach to generate binding partners that interact specifically with the light state or the dark state conformation of two light-switchable proteins: PYP, a test case for a protein with no known partners, and AsLOV2 a well-characterized protein. We show that these novel light-switchable protein-protein interactions can function in living cells to control subcellular localization processes.
4.

A Single-Component Optogenetic System Allows Stringent Switch of Gene Expression in Yeast Cells.

blue CRY2/CIB1 VVD S. cerevisiae Cell cycle control Transgene expression
ACS Synth Biol, 4 Sep 2018 DOI: 10.1021/acssynbio.8b00180 Link to full text
Abstract: Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔN Sic1 genes, respectively. Furthermore, we engineered a bidirectional expression module that allows the simultaneous control of the expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative, and spatially specific degradation of ssrA-tagged protein was observed. We suggest that this single-component optogenetic system will be immensely helpful in understanding cellular gene regulatory networks and in the design of robust genetic circuits for synthetic biology.
5.

Optical activation of TrkA signaling.

blue CRY2/CIB1 CRY2/CRY2 NIH/3T3 PC-12 Signaling cascade control Cell differentiation
ACS Synth Biol, 5 Jul 2018 DOI: 10.1021/acssynbio.8b00126 Link to full text
Abstract: Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 (CRY2) and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports the survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.
6.

Reversible Social Self-Sorting of Colloidal Cell-Mimics with Blue Light Switchable Proteins.

blue iLID Magnets in vitro
ACS Synth Biol, 21 Jun 2018 DOI: 10.1021/acssynbio.8b00250 Link to full text
Abstract: Towards the bottom-up assembly of synthetic cells from molecular building blocks it is an ongoing challenge to assemble micrometer sized compartments that host different processes into precise multicompartmental assemblies, also called prototissues. The difficulty lies in controlling interactions between different compartments dynamically both in space and time, as these interactions determine how they organize with respect to each other and how they work together. In this study, we have been able to control the self-assembly and social self-sorting of four different types of colloids, which we use as a model for synthetic cells, into two separate families with visible light. For this purpose we used two photoswitchable protein pairs (iLID/Nano and nHagHigh/pMagHigh) that both reversibly heterodimerize upon blue light exposure and dissociate from each other in the dark. These photoswitchable proteins provide non-invasive, dynamic and reversible remote control under biocompatible conditions over the self-assembly process with unprecedented spatial and temporal precision. In addition, each protein pair brings together specifically two different types of colloids. The orthogonality of the two protein pairs enables social self-sorting of a four component mixture into two distinct families of colloidal aggregates with controlled arrangements. These results will ultimately pave the way for the bottom-up assembly of multicompartment synthetic prototissues of a higher complexity, enabling us to control precisely and dynamically the organization of different compartments in space and time.
7.

OptoBase: A web platform for molecular optogenetics.

ACS Synth Biol, 18 Jun 2018 DOI: 10.1021/acssynbio.8b00120 Link to full text
Abstract: OptoBase is an online platform for molecular optogenetics. At its core is a hand-annotated and ontology-supported database that aims to cover all existing optogenetic switches and publications, which is further complemented with a collection of convenient optogenetics-related web tools. OptoBase is meant for both expert optogeneticists, to easily keep track of the field, as well as for all researchers who find optogenetics inviting as a powerful tool to address their biological questions of interest. It is available at https://www.optobase.org. This work also presents OptoBase-based analysis of the trends in molecular optogenetics.
8.

Optogenetic inhibition of Gαq protein signaling reduces calcium oscillation stochasticity.

blue CRY2/CIB1 HEK293T Signaling cascade control Immediate control of second messengers
ACS Synth Biol, 24 May 2018 DOI: 10.1021/acssynbio.8b00065 Link to full text
Abstract: As fast terminators of G-protein coupled receptor (GPCR) signaling, regulators of G-protein signaling (RGS) serve critical roles in fine-tuning second messenger levels and, consequently, cellular responses to external stimuli. Here, we report the creation of an optogenetic RGS2 (opto-RGS2) that suppresses agonist-evoked calcium oscillations by the inactivation of Gαq protein. In this system, cryptochrome-mediated hetero-dimerization of the catalytic RGS2-box with its N-terminal amphipathic helix reconstitutes a functional membrane-localized complex that can dynamically suppress store-operated release of calcium. Engineered opto-RGS2 cell lines were used to establish the role of RGS2 as a key inhibitory feedback regulator of the stochasticity of the Gαq-mediated calcium spike timing. RGS2 reduced the stochasticity of carbachol-stimulated calcium oscillations, and the feedback inhibition was coupled to the global calcium elevation by calmodulin/RGS2 interactions. The identification of a critical negative feedback circuit exemplifies the utility of optogenetic approaches for interrogating RGS/GPCR biology and calcium encoding principles through temporally precise molecular gain-of-function.
9.

Bioprinting Living Biofilms through Optogenetic Manipulation.

blue red BlrP1 BphS P. aeruginosa Control of cell-cell / cell-material interactions Immediate control of second messengers Multichromatic
ACS Synth Biol, 18 Apr 2018 DOI: 10.1021/acssynbio.8b00003 Link to full text
Abstract: In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living bacteria to autonomically form patterned biofilms following prescribed illumination. The results showed that through optogenetic manipulation, patterned bacterial communities with high spatial resolution (approximately 10 μm) could be constructed in 6 h. Thus, optogenetic manipulation greatly increases the range of available bioprinting techniques.
10.

A green light-responsive system for the control of transgene expression in mammalian and plant cells.

green TtCBD A. thaliana leaf protoplasts Cos-7 HEK293 HeLa NIH/3T3
ACS Synth Biol, 10 Apr 2018 DOI: 10.1021/acssynbio.7b00450 Link to full text
Abstract: The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH6 and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene expression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.
11.

Cell-free optogenetic gene expression system.

blue EL222 in vitro
ACS Synth Biol, 29 Mar 2018 DOI: 10.1021/acssynbio.7b00422 Link to full text
Abstract: Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, its vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modelling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of target gene to generate oscillatory waveform. Deterministic model developed was able to recapitulate the system behaviour and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.
12.

Rewiring Calcium Signaling for Precise Transcriptional Reprogramming.

blue AsLOV2 LOVTRAP HEK293T HeLa Endogenous gene expression Immediate control of second messengers
ACS Synth Biol, 6 Mar 2018 DOI: 10.1021/acssynbio.7b00467 Link to full text
Abstract: Tools capable of modulating gene expression in living organisms are very useful for interrogating the gene regulatory network and controlling biological processes. The catalytically inactive CRISPR/Cas9 (dCas9), when fused with repressive or activating effectors, functions as a versatile platform to reprogram gene transcription at targeted genomic loci. However, without temporal control, the application of these reprogramming tools will likely cause off-target effects and lack strict reversibility. To overcome this limitation, we report herein the development of a chemical or light-inducible transcriptional reprogramming device that combines photoswitchable genetically encoded calcium actuators with dCas9 to control gene expression. By fusing an engineered Ca2+-responsive NFAT fragment with dCas9 and transcriptional coactivators, we harness the power of light to achieve photoinducible transcriptional reprogramming in mammalian cells. This synthetic system (designated CaRROT) can also be used to document calcium-dependent activity in mammals after exposure to ligands or chemicals that would elicit calcium response inside cells.
13.

Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics.

red PhyB/PIF3 HEK293 HeLa Huh-7 NIH/3T3
ACS Synth Biol, 4 Jan 2018 DOI: 10.1021/acssynbio.7b00413 Link to full text
Abstract: Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics, is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and for the first time, combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.
14.

Spatiotemporal Control of TGF-β Signaling with Light.

blue CRY2/CIB1 HeLa Signaling cascade control
ACS Synth Biol, 14 Dec 2017 DOI: 10.1021/acssynbio.7b00225 Link to full text
Abstract: Cells employ signaling pathways to make decisions in response to changes in their immediate environment. Transforming growth factor beta (TGF-β) is an important growth factor that regulates many cellular functions in development and disease. Although the molecular mechanisms of TGF-β signaling have been well studied, our understanding of this pathway is limited by the lack of tools that allow the control of TGF-β signaling with high spatiotemporal resolution. Here, we developed an optogenetic system (optoTGFBRs) that enables the precise control of TGF-β signaling in time and space. Using the optoTGFBRs system, we show that TGF-β signaling can be selectively and sequentially activated in single cells through the modulation of the pattern of light stimulations. By simultaneously monitoring the subcellular localization of TGF-β receptor and Smad2 proteins, we characterized the dynamics of TGF-β signaling in response to different patterns of blue light stimulations. The spatial and temporal precision of light control will make the optoTGFBRs system as a powerful tool for quantitative analyses of TGF-β signaling at the single cell level.
15.

Optogenetic Control of Endoplasmic Reticulum-Mitochondria Tethering.

blue near-infrared BphP1/Q-PAS1 FKF1/GI iLID Magnets HEK293T NIH/3T3 primary mouse cortical neurons Organelle manipulation
ACS Synth Biol, 4 Dec 2017 DOI: 10.1021/acssynbio.7b00248 Link to full text
Abstract: The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.
16.

Engineering an E. coli Near-Infrared Light Sensor.

near-infrared BphP1/PpsR2 E. coli
ACS Synth Biol, 9 Nov 2017 DOI: 10.1021/acssynbio.7b00289 Link to full text
Abstract: Optogenetics is a technology wherein researchers combine light and genetically engineered photoreceptors to control biological processes with unrivaled precision. Near-infrared (NIR) wavelengths (>700 nm) are desirable optogenetic inputs due to their low phototoxicity and spectral isolation from most photoproteins. The bacteriophytochrome photoreceptor 1 (BphP1), found in several purple photosynthetic bacteria, senses NIR light and activates transcription of photosystem promoters by binding to and inhibiting the transcriptional repressor PpsR2. Here, we examine the response of a library of output promoters to increasing levels of Rhodopseudomonas palustris PpsR2 expression, and we identify that of Bradyrhizobium sp. BTAi1 crtE as the most strongly repressed in Escherichia coli. Next, we optimize Rps. palustris bphP1 and ppsR2 expression in a strain engineered to produce the required chromophore biliverdin IXα in order to demonstrate NIR-activated transcription. Unlike a previously engineered bacterial NIR photoreceptor, our system does not require production of a second messenger, and it exhibits rapid response dynamics. It is also the most red-shifted bacterial optogenetic tool yet reported by approximately 50 nm. Accordingly, our BphP1-PpsR2 system has numerous applications in bacterial optogenetics.
17.

Optogenetics Manipulation Enables Prevention of Biofilm Formation of Engineered Pseudomonas aeruginosa on Surfaces.

blue YtvA P. aeruginosa Transgene expression Control of cell-cell / cell-material interactions
ACS Synth Biol, 31 Oct 2017 DOI: 10.1021/acssynbio.7b00273 Link to full text
Abstract: Synthetic biologists have attempted to solve real-world problems, such as those of bacterial biofilms, that are involved in the pathogenesis of many clinical infections and difficult to eliminate. To address this, we employed a blue light responding system and integrated it into the chromosomes of Pseudomonas aeruginosa. With making rational adaptions and improvements of the light-activated system, we provided a robust and convenient means to spatiotemporally control gene expression and manipulate biological processes with minimal perturbation in P. aeruginosa. It increased the light-induced gene expression up to 20-fold. Moreover, we deliberately introduced a functional protein gene PA2133 containing an EAL domain to degrade c-di-GMP into the modified system, and showed that the optimally engineered optogenetic tool inhibited the formation of P. aeruginosa biofilms through the induction of blue light, resulting in much sparser and thinner biofilms. Our approach establishes a methodology for leveraging the tools of synthetic biology to guide biofilm formation and engineer biofilm patterns with unprecedented spatiotemporal resolution. Furthermore, the findings suggest that the synthetic optogenetic system may provide a promising strategy that could be applied to control and fight biofilms.
18.

Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control.

blue Magnets E. coli
ACS Synth Biol, 18 Oct 2017 DOI: 10.1021/acssynbio.7b00169 Link to full text
Abstract: Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.
19.

Blue Light Switchable Bacterial Adhesion as a Key Step toward the Design of Biofilms.

blue Magnets E. coli in vitro Control of cell-cell / cell-material interactions
ACS Synth Biol, 17 Aug 2017 DOI: 10.1021/acssynbio.7b00197 Link to full text
Abstract: The control of where and when bacteria adhere to a substrate is a key step toward controlling the formation and organization in biofilms. This study shows how we engineer bacteria to adhere specifically to substrates with high spatial and temporal control under blue light, but not in the dark, by using photoswitchable interaction between nMag and pMag proteins. For this, we express pMag proteins on the surface of E. coli so that the bacteria can adhere to substrates with immobilized nMag protein under blue light. These adhesions are reversible in the dark and can be repeatedly turned on and off. Further, the number of bacteria that can adhere to the substrate as well as the attachment and detachment dynamics are adjustable by using different point mutants of pMag and altering light intensity. Overall, the blue light switchable bacteria adhesions offer reversible, tunable and bioorthogonal control with exceptional spatial and temporal resolution. This enables us to pattern bacteria on substrates with great flexibility.
20.

An Engineered Optogenetic Switch for Spatiotemporal Control of Gene Expression, Cell Differentiation, and Tissue Morphogenesis.

blue CRY2/CIB1 C3H/10T1/2 HEK293T mouse in vivo Transgene expression Cell differentiation Developmental processes Nucleic acid editing
ACS Synth Biol, 9 Aug 2017 DOI: 10.1021/acssynbio.7b00147 Link to full text
Abstract: The precise spatial and temporal control of gene expression, cell differentiation, and tissue morphogenesis has widespread application in regenerative medicine and the study of tissue development. In this work, we applied optogenetics to control cell differentiation and new tissue formation. Specifically, we engineered an optogenetic "on" switch that provides permanent transgene expression following a transient dose of blue light illumination. To demonstrate its utility in controlling cell differentiation and reprogramming, we incorporated an engineered form of the master myogenic factor MyoD into this system in multipotent cells. Illumination of cells with blue light activated myogenic differentiation, including upregulation of myogenic markers and fusion into multinucleated myotubes. Cell differentiation was spatially patterned by illumination of cell cultures through a photomask. To demonstrate the application of the system to controlling in vivo tissue development, the light inducible switch was used to control the expression of VEGF and angiopoietin-1, which induced angiogenic sprouting in a mouse dorsal window chamber model. Live intravital microscopy showed illumination-dependent increases in blood-perfused microvasculature. This optogenetic switch is broadly useful for applications in which sustained and patterned gene expression is desired following transient induction, including tissue engineering, gene therapy, synthetic biology, and fundamental studies of morphogenesis.
21.

Mini Photobioreactors for in Vivo Real-Time Characterization and Evolutionary Tuning of Bacterial Optogenetic Circuit.

green CcaS/CcaR E. coli
ACS Synth Biol, 5 Jun 2017 DOI: 10.1021/acssynbio.7b00091 Link to full text
Abstract: The current standard protocols for characterizing the optogenetic circuit of bacterial cells using flow cytometry in light tubes and light exposure of culture plates are tedious, labor-intensive, and cumbersome. In this work, we engineer a bioreactor with working volume of ∼10 mL for in vivo real-time optogenetic characterization of E. coli with a CcaS-CcaR light-sensing system. In the bioreactor, optical density measurements, reporter protein fluorescence detection, and light input stimuli are provided by four light-emitting diode sources and two photodetectors. Once calibrated, the device can cultivate microbial cells and record their growth and gene expression without human intervention. We measure gene expression during cell growth with different organic substrates (glucose, succinate, acetate, pyruvate) as carbon sources in minimal medium and demonstrate evolutionary tuning of the optogenetic circuit by serial dilution passages.
22.

A Phytochrome-Derived Photoswitch for Intracellular Transport.

blue red PhyB/PIF6 TULIP Cos-7 U-2 OS Organelle manipulation Multichromatic
ACS Synth Biol, 30 Mar 2017 DOI: 10.1021/acssynbio.6b00333 Link to full text
Abstract: Cells depend on the proper positioning of their organelles, suggesting that active manipulation of organelle positions can be used to explore spatial cell biology and to restore cellular defects caused by organelle misplacement. Recently, blue-light dependent recruitment of specific motors to selected organelles has been shown to alter organelle motility and positioning, but these approaches lack rapid and active reversibility. The light-dependent interaction of phytochrome B with its interacting factors has been shown to function as a photoswitch, dimerizing under red light and dissociating under far-red light. Here we engineer phytochrome domains into photoswitches for intracellular transport that enable the reversible interaction between organelles and motor proteins. Using patterned illumination and live-cell imaging, we demonstrate that this system provides unprecedented spatiotemporal control. We also demonstrate that it can be used in combination with a blue-light dependent system to independently control the positioning of two different organelles. Precise optogenetic control of organelle motility and positioning will provide a better understanding of and control over the spatial biology of cells.
23.

Assembly Domain-Based Optogenetic System for the Efficient Control of Cellular Signaling.

blue Magnets Cos-7 HEK293T Control of cytoskeleton / cell motility / cell shape
ACS Synth Biol, 3 Mar 2017 DOI: 10.1021/acssynbio.7b00022 Link to full text
Abstract: We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein-protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca(2+)/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation.
24.

Engineered Photoactivatable Genetic Switches Based on the Bacterium Phage T7 RNA Polymerase.

blue Magnets VVD E. coli
ACS Synth Biol, 15 Nov 2016 DOI: 10.1021/acssynbio.6b00248 Link to full text
Abstract: Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains. For the best split position found, working switches exploit either the light-induced interactions between the VVD domains or allosteric effects. The split fragments show high modularity when they are combined with different regulatory domains such as those with chemically inducible interaction, enabling chemically controlled switches. To summarize, the T7 RNA polymerase-based switches are powerful tools to implement light-activated gene expression in different contexts. Moreover, results about the studied split positions and domain organizations may facilitate future engineering studies on this and on related proteins.
25.

Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli.

violet UirS/UirR E. coli
ACS Synth Biol, 11 May 2016 DOI: 10.1021/acssynbio.6b00068 Link to full text
Abstract: We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS UirS-UirR from Synechocystis PCC6803 to E. coli. We demonstrate that the promoter of the small RNA csiR1, previously shown to be activated by inorganic carbon stress, is a UirS-UirR output. Additionally, in contrast to a recently proposed sequestration model, we show that the sensor histidine kinase UirS phosphorylates the response regulator UirR to activate PcsiR1 transcription in response to UV-violet light. Finally, we measure changes in UirS-UirR output minutes after a change in light input and exploit these rapid dynamics to program a challenging gene expression signal with high predictability. UirS-UirR is the first engineered transcriptional regulatory tool activated exclusively by UV-violet light, and the most blue shifted photoreversible transcriptional regulatory tool.
Submit a new publication to our database