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 42 results

Optoregulated Drug Release from an Engineered Living Material: Self-Replenishing Drug Depots for Long-Term, Light-Regulated Delivery.

blue YtvA E. coli Transgene expression
Small, 27 Dec 2018 DOI: 10.1002/smll.201804717 Link to full text
Abstract: On-demand and long-term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self-replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin-free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light-regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.

Engineering a light-responsive, quorum quenching biofilm to mitigate biofouling on water purification membranes.

blue red BphS EB1 E. coli Control of cell-cell / cell-material interactions Immediate control of second messengers Multichromatic
Sci Adv, 7 Dec 2018 DOI: 10.1126/sciadv.aau1459 Link to full text
Abstract: Quorum quenching (QQ) has been reported to be a promising approach for membrane biofouling control. Entrapment of QQ bacteria in porous matrices is required to retain them in continuously operated membrane processes and to prevent uncontrollable biofilm formation by the QQ bacteria on membrane surfaces. It would be more desirable if the formation and dispersal of biofilms by QQ bacteria could be controlled so that the QQ bacterial cells are self-immobilized, but the QQ biofilm itself still does not compromise membrane performance. In this study, we engineered a QQ bacterial biofilm whose growth and dispersal can be modulated by light through a dichromatic, optogenetic c-di-GMP gene circuit in which the bacterial cells sense near-infrared (NIR) light and blue light to adjust its biofilm formation by regulating the c-di-GMP level. We also demonstrated the potential application of the engineered light-responsive QQ biofilm in mitigating biofouling of water purification forward osmosis membranes. The c-di-GMP-targeted optogenetic approach for controllable biofilm development we have demonstrated here should prove widely applicable for designing other controllable biofilm-enabled applications such as biofilm-based biocatalysis.

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.

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43.

blue YtvA E. coli Transgene expression Control of cell-cell / cell-material interactions
J Vis Exp, 23 Oct 2018 DOI: 10.3791/58625 Link to full text
Abstract: Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct-pDawn-Ag43-that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 μm can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

A light-controlled cell lysis system in bacteria.

blue YtvA E. coli Transgene expression Cell death
J Ind Microbiol Biotechnol, 8 May 2018 DOI: 10.1007/s10295-018-2034-4 Link to full text
Abstract: Intracellular products (e.g., insulin), which are obtained through cell lysis, take up a big share of the biotech industry. It is often time-consuming, laborious, and environment-unfriendly to disrupt bacterial cells with traditional methods. In this study, we developed a molecular device for controlling cell lysis with light. We showed that intracellular expression of a single lysin protein was sufficient for efficient bacterial cell lysis. By placing the lysin-encoding gene under the control of an improved light-controlled system, we successfully controlled cell lysis by switching on/off light: OD600 of the Escherichia coli cell culture was decreased by twofold when the light-controlled system was activated under dark condition. We anticipate that our work would not only pave the way for cell lysis through a convenient biological way in fermentation industry, but also provide a paradigm for applying the light-controlled system in other fields of biotech industry.

Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells.

violet cPAC E. coli in vitro Immediate control of second messengers
J Biol Chem, 9 Apr 2018 DOI: 10.1074/jbc.ra118.002258 Link to full text
Abstract: Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl-cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in non-photosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleussp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibited a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells. tissues, and whole organisms with light across the visible spectrum and into near IR.

Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression.

blue YtvA E. coli Transgene expression Control of cell-cell / cell-material interactions
Proc Natl Acad Sci USA, 19 Mar 2018 DOI: 10.1073/pnas.1720676115 Link to full text
Abstract: Bacterial biofilms represent a promising opportunity for engineering of microbial communities. However, our ability to control spatial structure in biofilms remains limited. Here we engineerEscherichia coliwith a light-activated transcriptional promoter (pDawn) to optically regulate expression of an adhesin gene (Ag43). When illuminated with patterned blue light, long-term viable biofilms with spatial resolution down to 25 μm can be formed on a variety of substrates and inside enclosed culture chambers without the need for surface pretreatment. A biophysical model suggests that the patterning mechanism involves stimulation of transiently surface-adsorbed cells, lending evidence to a previously proposed role of adhesin expression during natural biofilm maturation. Overall, this tool-termed "Biofilm Lithography"-has distinct advantages over existing cell-depositing/patterning methods and provides the ability to grow structured biofilms, with applications toward an improved understanding of natural biofilm communities, as well as the engineering of living biomaterials and bottom-up approaches to microbial consortia design.

Optogenetic Control by Pulsed Illumination.

blue YtvA E. coli
Chembiochem, 14 Feb 2018 DOI: 10.1002/cbic.201800030 Link to full text
Abstract: Sensory photoreceptors evoke numerous adaptive responses in Nature and serve as light-gated actuators in optogenetics to enable the spatiotemporally precise, reversible and noninvasive control of cellular events. The output of optogenetic circuits can often be dialed in by varying illumination quality, quantity and duration. Here, we devise a programmable matrix of light-emitting diodes to efficiently probe the response of optogenetic systems to intermittently applied light of varying intensity and pulse frequency. Circuits for light-regulated gene expression markedly differed in their responses to pulsed illumination of a single color which sufficed for sequentially triggering them. In addition to quantity and quality, the pulse frequency of intermittent light hence provides a further input variable for output control in optogenetics and photobiology. Pulsed illumination schemes allow the reduction of overall light dose and facilitate the multiplexing of several light-dependent actuators and reporters.

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.

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.

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.

Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell.

blue YtvA E. coli Transgene expression
BMC Biotechnol, 7 Nov 2017 DOI: 10.1186/s12896-017-0402-1 Link to full text
Abstract: β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance.

Re-engineering the two-component systems as light-regulated in Escherichia coli.

red Cph1 E. coli
J Biosci, 20 Oct 2017 DOI: 10.1007/s12038-017-9711-8 Link to full text
Abstract: Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make use of various sensor-response circuits, called two-component systems (TCSs). A TCS comprises a histidine protein kinase (HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two components are coupled via a phosphorylation control mechanism. In a recent study, we adopted an optogenetics approach to re-engineer the sensor HKs in Escherichia coli as a light-sensing fusion protein. We constructed a light-controllable HK by replacing the original signal-specific sensing domain of HK with the light-sensing domain of Cph1 from Cyanobacteria Synechocystis, so that HK can be investigated by red light. Here, we extended the study to other 16 HK-RR TCSs and constructed a library of light-responsible HK-Cph1 chimeras. By taking the NarX-NarL system as an example, we demonstrated the light responsiveness of the constructed chimera and investigated the frequency response of the NarXNarL system. The constructed library serves as a toolkit for future TCS study using optogenetics approach.

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.

Using Light-Activated Enzymes for Modulating Intracellular c-di-GMP Levels in Bacteria.

blue red BphS EB1 A. brasilense E. coli Multichromatic
Methods Mol Biol, 10 Sep 2017 DOI: 10.1007/978-1-4939-7240-1_14 Link to full text
Abstract: Signaling pathways involving second messenger c-di-GMP regulate various aspects of bacterial physiology and behavior. We describe the use of a red light-activated diguanylate cyclase (c-di-GMP synthase) and a blue light-activated c-di-GMP phosphodiesterase (hydrolase) for manipulating intracellular c-di-GMP levels in bacterial cells. We illustrate the application of these enzymes in regulating several c-di-GMP-dependent phenotypes, i.e., motility and biofilm phenotypes in E. coli and chemotactic behavior in the alphaproteobacterium Azospirillum brasilense. We expect these light-activated enzymes to be also useful in regulating c-di-GMP-dependent processes occurring at the fast timescale, for spatial control of bacterial populations, as well as for analyzing c-di-GMP-dependent phenomena at the single-cell level.

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.

Photocontrolled reversible self-assembly of dodecamer nitrilase.

blue iLID E. coli in vitro
Bioresour Bioprocess, 4 Aug 2017 DOI: 10.1186/s40643-017-0167-3 Link to full text
Abstract: Naturally photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer. In this study, a method for the precise and reversible inducible self-assembly of dodecamer nitrilase in vivo (in Escherichia coli) and in vitro (in a cell-free solution) was developed by means of the photoswitch-improved light-inducible dimer (iLID) system which could induce protein-protein dimerization.

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.

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.

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.

Optogenetic Module for Dichromatic Control of c-di-GMP Signaling.

blue red BphS EB1 E. coli in vitro Immediate control of second messengers Multichromatic
J Bacteriol, 20 Mar 2017 DOI: 10.1128/jb.00014-17 Link to full text
Abstract: Many aspects of bacterial physiology and behavior including motility, surface attachment, and cell cycle, are controlled by the c-di-GMP-dependent signaling pathways on the scale of seconds-to-minutes. Interrogation of such processes in real time requires tools for introducing rapid and reversible changes in intracellular c-di-GMP levels. Inducing expression of genes encoding c-di-GMP synthetic (diguanylate cyclases) and degrading (c-di-GMP phosphodiesterase) enzymes by chemicals may not provide adequate temporal control. In contrast, light-controlled diguanylate cyclases and phosphodiesterases can be quickly activated and inactivated. A red/near-infrared light-regulated diguanylate cyclase, BphS, has been engineered earlier, yet a complementary light-activated c-di-GMP phosphodiesterase has been lacking. In search of such a phosphodiesterase, we investigated two homologous proteins from Allochromatium vinosum and Magnetococcus marinus, designated BldP, which contain C-terminal EAL-BLUF modules, where EAL is a c-di-GMP phosphodiesterase domain and BLUF is a blue light sensory domain. Characterization of the BldP proteins in Escherichia coli and in vitro showed that they possess light-activated c-di-GMP phosphodiesterase activities. Interestingly, light activation in both enzymes was dependent on oxygen levels. The truncated EAL-BLUF fragment from A. vinosum BldP lacked phosphodiesterase activity, whereas a similar fragment from M. marinus BldP, designated EB1, possessed such activity that was highly (>30-fold) upregulated by light. Following light withdrawal, EB1 reverted to the inactive ground state with a half-life of ∼6 min. Therefore, the blue light-activated phosphodiesterase, EB1, can be used in combination with the red/near-infrared light-regulated diguanylate cyclase, BphS, for bidirectional regulation of c-di-GMP-dependent processes in E. coli as well as other bacterial and nonbacterial cells.IMPORTANCE Regulation of motility, attachment to surfaces, cell cycle, and other bacterial processes controlled by the c-di-GMP signaling pathways occurs at a fast (seconds-to-minutes) pace. Interrogating these processes at high temporal and spatial resolution using chemicals is difficult-to-impossible, while optogenetic approaches may prove useful. We identified and characterized a robust, blue light-activated c-di-GMP phosphodiesterase (hydrolase) that complements a previously engineered red/near-infrared light-regulated diguanylate cyclase (c-di-GMP synthase). These two enzymes form a dichromatic module for manipulating intracellular c-di-GMP levels in bacterial and nonbacterial cells.

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.

An open-hardware platform for optogenetics and photobiology.

blue green red CcaS/CcaR CRY2/CIB1 PhyB/PIF6 E. coli HeLa S. cerevisiae
Sci Rep, 2 Nov 2016 DOI: 10.1038/srep35363 Link to full text
Abstract: In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.

Engineering of temperature- and light-switchable Cas9 variants.

blue RsLOV E. coli in vitro
Nucleic Acids Res, 15 Oct 2016 DOI: 10.1093/nar/gkw930 Link to full text
Abstract: Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.

Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth.

green CcaS/CcaR E. coli
Nat Commun, 26 Aug 2016 DOI: 10.1038/ncomms12546 Link to full text
Abstract: Dynamic control of gene expression can have far-reaching implications for biotechnological applications and biological discovery. Thanks to the advantages of light, optogenetics has emerged as an ideal technology for this task. Current state-of-the-art methods for optical expression control fail to combine precision with repeatability and cannot withstand changing operating culture conditions. Here, we present a novel fully automatic experimental platform for the robust and precise long-term optogenetic regulation of protein production in liquid Escherichia coli cultures. Using a computer-controlled light-responsive two-component system, we accurately track prescribed dynamic green fluorescent protein expression profiles through the application of feedback control, and show that the system adapts to global perturbations such as nutrient and temperature changes. We demonstrate the efficacy and potential utility of our approach by placing a key metabolic enzyme under optogenetic control, thus enabling dynamic regulation of the culture growth rate with potential applications in bacterial physiology studies and biotechnology.
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