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 41 results

Diverse light responses of cyanobacteria mediated by phytochrome superfamily photoreceptors.

green red violet Cyanobacteriochromes Phytochromes Review
Nat Rev Microbiol, Jan 2019 DOI: 10.1038/s41579-018-0110-4 Link to full text
Abstract: Cyanobacteria are an evolutionarily and ecologically important group of prokaryotes. They exist in diverse habitats, ranging from hot springs and deserts to glaciers and the open ocean. The range of environments that they inhabit can be attributed in part to their ability to sense and respond to changing environmental conditions. As photosynthetic organisms, one of the most crucial parameters for cyanobacteria to monitor is light. Cyanobacteria can sense various wavelengths of light and many possess a range of bilin-binding photoreceptors belonging to the phytochrome superfamily. Vital cellular processes including growth, phototaxis, cell aggregation and photosynthesis are tuned to environmental light conditions by these photoreceptors. In this Review, we examine the physiological responses that are controlled by members of this diverse family of photoreceptors and discuss the signal transduction pathways through which these photoreceptors operate. We highlight specific examples where the activities of multiple photoreceptors function together to fine-tune light responses. We also discuss the potential application of these photosensing systems in optogenetics and synthetic biology.

Using Synthetic Biology to Engineer Spatial Patterns.

blue green red Cryptochromes Cyanobacteriochromes LOV domains Phytochromes Review
Adv Biosyst, 17 Dec 2018 DOI: 10.1002/adbi.201800280 Link to full text
Abstract: Synthetic biology has emerged as a multidisciplinary field that provides new tools and approaches to address longstanding problems in biology. It integrates knowledge from biology, engineering, mathematics, and biophysics to build—rather than to simply observe and perturb—biological systems that emulate natural counterparts or display novel properties. The interface between synthetic and developmental biology has greatly benefitted both fields and allowed to address questions that would remain challenging with classical approaches due to the intrinsic complexity and essentiality of developmental processes. This Progress Report provides an overview of how synthetic biology can help to understand a process that is crucial for the development of multicellular organisms: pattern formation. It reviews the major mechanisms of genetically encoded synthetic systems that have been engineered to establish spatial patterns at the population level. Limitations, challenges, applications, and potential opportunities of synthetic pattern formation are also discussed.

Programming Bacteria With Light—Sensors and Applications in Synthetic Biology

blue cyan green near-infrared red UV violet Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Front Microbiol, 8 Nov 2018 DOI: 10.3389/fmicb.2018.02692 Link to full text
Abstract: Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture.

Optogenetic Medicine: Synthetic Therapeutic Solutions Precision-Guided by Light.

blue cyan green near-infrared red UV Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Cold Spring Harb Perspect Med, 5 Oct 2018 DOI: 10.1101/cshperspect.a034371 Link to full text
Abstract: Gene- and cell-based therapies are well recognized as central pillars of next-generation medicine, but controllability remains a critical issue for clinical applications. In this context, optogenetics is opening up exciting new opportunities for precision-guided medicine by using illumination with light of appropriate intensity and wavelength as a trigger signal to achieve pinpoint spatiotemporal control of cellular activities, such as transgene expression. In this review, we highlight recent advances in optogenetics, focusing on devices for biomedical applications. We introduce the construction and applications of optogenetic-based biomedical tools to treat neurological diseases, diabetes, heart diseases, and cancer, as well as bioelectronic implants that combine light-interfaced electronic devices and optogenetic systems into portable personalized precision bioelectronic medical tools. Optogenetics-based technology promises the capability to achieve traceless, remotely controlled precision dosing of an enormous range of therapeutic outputs. Finally, we discuss the prospects for optogenetic medicine, as well as some emerging challenges.

Synthetic Light-Activated Ion Channels for Optogenetic Activation and Inhibition.

blue green BeCyclOp (BeGC1) bPAC (BlaC) D. melanogaster in vivo rat hippocampal neurons Xenopus oocytes Immediate control of second messengers Neuronal activity control
Front Neurosci, 2 Oct 2018 DOI: 10.3389/fnins.2018.00643 Link to full text
Abstract: Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2+) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca2+ might be desirable. Moreover, there is need for an efficient light-gated potassium (K+) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca2+ and K+ in cell physiology, light-activated Ca2+-permeant and K+-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca2+-permeant and K+-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca2+ or for K+, respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca2+-permeant channel, and to body extension when expressing the light-sensitive K+ channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.

Light‐Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology.

blue cyan green near-infrared red UV BLUF domains Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Adv Sci, 30 Sep 2018 DOI: 10.1002/advs.201800952 Link to full text
Abstract: The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology‐inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light‐controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non‐neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light‐sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light‐controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.

Plasticity in oligomerization, operator architecture, and DNA binding in the mode of action of a bacterial B12-based photoreceptor.

green Cobalamin-binding domains Background
J Biol Chem, 27 Sep 2018 DOI: 10.1074/jbc.ra118.004838 Link to full text
Abstract: Newly discovered bacterial photoreceptors called CarH sense light by using 5'-deoxyadenosylcobalamin (AdoCbl). They repress their own expression and that of genes for carotenoid synthesis by binding in the dark to operator DNA as AdoCbl-bound tetramers, whose light-induced disassembly relieves repression. High-resolution structures of Thermus thermophilus CarHTt have provided snapshots of the dark and light states and have revealed a unique DNA-binding mode whereby only three out of four DNA binding domains contact an operator comprising three tandem direct repeats. To gain further insights into CarH photoreceptors and employing biochemical, spectroscopic, mutational and computational analyses, here we investigated CarHBm from Bacillus megaterium We found that apoCarHBm, unlike monomeric apoCarHTt, is an oligomeric molten globule that forms DNA-binding tetramers in the dark only upon AdoCbl binding, which requires a conserved W-x9-EH motif. Light relieved DNA binding by disrupting CarHBm tetramers to dimers, rather than to monomers as with CarHTt CarHBm operators resembled that of CarHTt, but were larger by one repeat and overlapped with the -35 or -10 promoter elements. This design persisted in a six-repeat, multipartite operator we discovered upstream of a gene encoding an Spx global redox-response regulator whose photoregulated expression links photooxidative and general redox responses in B. megaterium Interestingly, CarHBm recognized the smaller CarHTt operator, revealing an adaptability possibly related to the linker bridging the DNA- and AdoCbl-binding domains. Our findings highlight a remarkable plasticity in the mode of action of B12-based CarH photoreceptors, important for their biological functions and development as optogenetic tools.

A compendium of chemical and genetic approaches to light-regulated gene transcription.

blue cyan green near-infrared red UV BLUF domains Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Crit Rev Biochem Mol Biol, 24 Jul 2018 DOI: 10.1080/10409238.2018.1487382 Link to full text
Abstract: On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.

Illuminating pathogen-host intimacy through optogenetics.

blue green red BLUF domains Cryptochromes LOV domains Opsins Phytochromes Review
PLoS Pathog, 12 Jul 2018 DOI: 10.1371/journal.ppat.1007046 Link to full text
Abstract: The birth and subsequent evolution of optogenetics has resulted in an unprecedented advancement in our understanding of the brain. Its outstanding success does usher wider applications; however, the tool remains still largely relegated to neuroscience. Here, we introduce selected aspects of optogenetics with potential applications in infection biology that will not only answer long-standing questions about intracellular pathogens (parasites, bacteria, viruses) but also broaden the dimension of current research in entwined models. In this essay, we illustrate how a judicious integration of optogenetics with routine methods can illuminate the host-pathogen interactions in a way that has not been feasible otherwise.

Blue-Light Receptors for Optogenetics.

blue green red UV BLUF domains Cryptochromes Fluorescent proteins LOV domains Opsins Phytochromes UV receptors Review
Chem Rev, 9 Jul 2018 DOI: 10.1021/acs.chemrev.8b00163 Link to full text
Abstract: Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Optogenetic regulation of transcription.

blue green near-infrared red Cryptochromes Cyanobacteriochromes LOV domains Phytochromes Review
BMC Neurosci, 19 Apr 2018 DOI: 10.1186/s12868-018-0411-6 Link to full text
Abstract: Optogenetics has become widely recognized for its success in real-time control of brain neurons by utilizing nonmammalian photosensitive proteins to open or close membrane channels. Here we review a less well known type of optogenetic constructs that employs photosensitive proteins to transduce the signal to regulate gene transcription, and its possible use in medicine. One of the problems with existing gene therapies is that they could remain active indefnitely while not allowing regulated transgene production on demand. Optogenetic regulation of transcription (ORT) could potentially be used to regulate the production of a biological drug in situ, by repeatedly applying light to the tissue, and inducing expression of therapeutic transgenes when needed. Red and near infrared wavelengths, which are capable of penetration into tissues, have potential for therapeutic applications. Existing ORT systems are reviewed herein with these considerations in mind.

Optogenetics: A Primer for Chemists.

blue green near-infrared red UV BLUF domains Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Chembiochem, 19 Apr 2018 DOI: 10.1002/cbic.201800013 Link to full text
Abstract: The field of optogenetics uses genetically encoded, light-responsive proteins to control physiological processes. This technology has been hailed as the one of the ten big ideas in brain science in the past decade,[1] the breakthrough of the decade,[2] and the method of the year in 2010[3] and again in 2014[4]. The excitement evidenced by these proclamations is confirmed by a couple of impressive numbers. The term "optogenetics" was coined in 2006.[5] As of December 2017, "optogenetics" is found in the title or abstract of almost 1600 currently funded National Institutes of Health grants. In addition, nearly 600 reviews on optogenetics have appeared since 2006, which averages out to approximately one review per week! However, in spite of these impressive numbers, the potential applications and implications of optogenetics are not even close to being fully realized. This is due, in large part, to the challenges associated with the design of optogenetic analogs of endogenous proteins. This review is written from a chemist's perspective, with a focus on the molecular strategies that have been developed for the construction of optogenetic proteins.

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.

Optogenetically controlled protein kinases for regulation of cellular signaling.

blue cyan green near-infrared red Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
Chem Soc Rev, 2 Mar 2018 DOI: 10.1039/c7cs00404d Link to full text
Abstract: Protein kinases are involved in the regulation of many cellular processes including cell differentiation, survival, migration, axon guidance and neuronal plasticity. A growing set of optogenetic tools, termed opto-kinases, allows activation and inhibition of different protein kinases with light. The optogenetic regulation enables fast, reversible and non-invasive manipulation of protein kinase activities, complementing traditional methods, such as treatment with growth factors, protein kinase inhibitors or chemical dimerizers. In this review, we summarize the properties of the existing optogenetic tools for controlling tyrosine kinases and serine-threonine kinases. We discuss how the opto-kinases can be applied for studies of spatial and temporal aspects of protein kinase signaling in cells and organisms. We compare approaches for chemical and optogenetic regulation of protein kinase activity and present guidelines for selection of opto-kinases and equipment to control them with light. We also describe strategies to engineer novel opto-kinases on the basis of various photoreceptors.

A miniaturized E. coli green light sensor with high dynamic range.

green CcaS/CcaR E. coli
Chembiochem, 8 Feb 2018 DOI: 10.1002/cbic.201800007 Link to full text
Abstract: Genetically-engineered photoreceptors enable unrivaled control over gene expression. Previously, we ported the Synechocystis PCC 6803 CcaSR two-component system, which is activated by green light and de-activated by red, into E. coli, resulting in a sensor with 6-fold dynamic range. Later, we optimized pathway protein expression levels and the output promoter sequence to decrease transcriptional leakiness and increase the dynamic range to approximately 120-fold. These CcaSR v1.0 and 2.0 systems have been used for precise quantitative, temporal, and spatial control of gene expression for a variety of applications. Recently, others have deleted two PAS domains of unknown function from the CcaS sensor histidine kinase in a CcaSR v1.0-like system. Here, we apply these deletions to CcaSR v2.0, resulting in a v3.0 light sensor with 4-fold lower leaky output and nearly 600-fold dynamic range. We demonstrate that the PAS domain deletions have no deleterious effect on CcaSR green light sensitivity or response dynamics. CcaSR v3.0 is the best performing engineered bacterial green light sensor available, and should have broad applications in fundamental and synthetic biology studies.

A novel optogenetically tunable frequency modulating oscillator.

green violet CcaS/CcaR UirS/UirR in silico
PLoS ONE, 1 Feb 2018 DOI: 10.1371/journal.pone.0183242 Link to full text
Abstract: Synthetic biology has enabled the creation of biological reconfigurable circuits, which perform multiple functions monopolizing a single biological machine; Such a system can switch between different behaviours in response to environmental cues. Previous work has demonstrated switchable dynamical behaviour employing reconfigurable logic gate genetic networks. Here we describe a computational framework for reconfigurable circuits in E.coli using combinations of logic gates, and also propose the biological implementation. The proposed system is an oscillator that can exhibit tunability of frequency and amplitude of oscillations. Further, the frequency of operation can be changed optogenetically. Insilico analysis revealed that two-component light systems, in response to light within a frequency range, can be used for modulating the frequency of the oscillator or stopping the oscillations altogether. Computational modelling reveals that mixing two colonies of E.coli oscillating at different frequencies generates spatial beat patterns. Further, we show that these oscillations more robustly respond to input perturbations compared to the base oscillator, to which the proposed oscillator is a modification. Compared to the base oscillator, the proposed system shows faster synchronization in a colony of cells for a larger region of the parameter space. Additionally, the proposed oscillator also exhibits lesser synchronization error in the transient period after input perturbations. This provides a strong basis for the construction of synthetic reconfigurable circuits in bacteria and other organisms, which can be scaled up to perform functions in the field of time dependent drug delivery with tunable dosages, and sets the stage for further development of circuits with synchronized population level behaviour.

Shaping bacterial population behavior through computer-interfaced control of individual cells.

green CcaS/CcaR E. coli
Nat Commun, 16 Nov 2017 DOI: 10.1038/s41467-017-01683-1 Link to full text
Abstract: Bacteria in groups vary individually, and interact with other bacteria and the environment to produce population-level patterns of gene expression. Investigating such behavior in detail requires measuring and controlling populations at the single-cell level alongside precisely specified interactions and environmental characteristics. Here we present an automated, programmable platform that combines image-based gene expression and growth measurements with on-line optogenetic expression control for hundreds of individual Escherichia coli cells over days, in a dynamically adjustable environment. This integrated platform broadly enables experiments that bridge individual and population behaviors. We demonstrate: (i) population structuring by independent closed-loop control of gene expression in many individual cells, (ii) cell-cell variation control during antibiotic perturbation, (iii) hybrid bio-digital circuits in single cells, and freely specifiable digital communication between individual bacteria. These examples showcase the potential for real-time integration of theoretical models with measurement and control of many individual cells to investigate and engineer microbial population behavior.

Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC.

green Opsins Background
J Biol Chem, 8 Nov 2017 DOI: 10.1074/jbc.m117.812685 Link to full text
Abstract: RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCCRho) and without (GCRho) the coiled-coil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GCRho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2',3'-dideoxyguanosine-5'-triphosphate, MnCl2, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzyme concentration. The low affinity observed for GCRho monomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

Applications of optobiology in intact cells and multi-cellular organisms.

blue cyan green near-infrared red Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
J Mol Biol, 4 Sep 2017 DOI: 10.1016/j.jmb.2017.08.015 Link to full text
Abstract: Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.

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.

B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release.

green TtCBD in vitro Control of cell-cell / cell-material interactions
Proc Natl Acad Sci USA, 22 May 2017 DOI: 10.1073/pnas.1621350114 Link to full text
Abstract: Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Engineering RGB color vision into Escherichia coli.

blue green red CcaS/CcaR Cph1 YtvA E. coli Multichromatic
Nat Chem Biol, 22 May 2017 DOI: 10.1038/nchembio.2390 Link to full text
Abstract: Optogenetic tools use colored light to rapidly control gene expression in space and time. We designed a genetically encoded system that gives Escherichia coli the ability to distinguish between red, green, and blue (RGB) light and respond by changing gene expression. We use this system to produce 'color photographs' on bacterial culture plates by controlling pigment production and to redirect metabolic flux by expressing CRISPRi guide RNAs.

Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii.

green Opsins Background
J Biol Chem, 4 May 2017 DOI: 10.1074/jbc.m117.789636 Link to full text
Abstract: RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one with red-shifted (D380E, λmax = 533 nm) absorption maxima relative to the wild-type protein (λmax = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

A photoconversion model for full spectral programming and multiplexing of optogenetic systems.

green red CcaS/CcaR Cph1 E. coli Multichromatic
Mol Syst Biol, 24 Apr 2017 DOI: 10.15252/msb.20167456 Link to full text
Abstract: Optogenetics combines externally applied light signals and genetically engineered photoreceptors to control cellular processes with unmatched precision. Here, we develop a mathematical model of wavelength- and intensity-dependent photoconversion, signaling, and output gene expression for our two previously engineered light-sensing Escherichia coli two-component systems. To parameterize the model, we develop a simple set of spectral and dynamical calibration experiments using our recent open-source "Light Plate Apparatus" device. In principle, the parameterized model should predict the gene expression response to any time-varying signal from any mixture of light sources with known spectra. We validate this capability experimentally using a suite of challenging light sources and signals very different from those used during the parameterization process. Furthermore, we use the model to compensate for significant spectral cross-reactivity inherent to the two sensors in order to develop a new method for programming two simultaneous and independent gene expression signals within the same cell. Our optogenetic multiplexing method will enable powerful new interrogations of how metabolic, signaling, and decision-making pathways integrate multiple input signals.

Green-Light-Induced Inactivation of Receptor Signaling Using Cobalamin-Binding Domains.

green MxCBD TtCBD HEK293 zebrafish in vivo Signaling cascade control Developmental processes
Angew Chem Int Ed Engl, 20 Mar 2017 DOI: 10.1002/anie.201611998 Link to full text
Abstract: Optogenetics and photopharmacology provide spatiotemporally precise control over protein interactions and protein function in cells and animals. Optogenetic methods that are sensitive to green light and can be used to break protein complexes are not broadly available but would enable multichromatic experiments with previously inaccessible biological targets. Herein, we repurposed cobalamin (vitamin B12) binding domains of bacterial CarH transcription factors for green-light-induced receptor dissociation. In cultured cells, we observed oligomerization-induced cell signaling for the fibroblast growth factor receptor 1 fused to cobalamin-binding domains in the dark that was rapidly eliminated upon illumination. In zebrafish embryos expressing fusion receptors, green light endowed control over aberrant fibroblast growth factor signaling during development. Green-light-induced domain dissociation and light-inactivated receptors will critically expand the optogenetic toolbox for control of biological processes.
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