We would like to thank Bal&#225;zsi lab for comments and suggestions, Dr. Karl P. Gerhardt and Dr. Jeffrey J. Tabor for helping us construct the first LPA, and Dr. Wilfried Weber for sharing the LOV2-degron plasmids. This work was supported by the National Institutes of Health [R35 GM122561 and T32 GM008444]; The Laufer Center for Physical and Quantitative Biology; and a National Defense Science and Engineering Graduate (NDSEG) Fellowship. Funding for open access charge: NIH [R35 GM122561].
Author contributions: M.T.G. and G.B. conceived the project. M.T.G., D.C., and L.G., performed the experiments. M.T.G., D.C., L.G., and G.B. analyzed the data and prepared the manuscript. G.B. and M.T.G. supervised the project.

The authors declare no competing financial interests.

Readers of this article can gain insight into the steps vital for characterizing optogenetic gene circuits (as well as other gene expression systems), including 1) gene circuit design, construction, and validation; 2) cell engineering for introducing gene circuits into stable cell lines (e.g., Flp-FRT recombination); 3) induction of the engineered cells with a light-based platform such as the LPA; 4) initial characterization of light induction assays via fluorescence microscopy; and 5) final gene expression characterization with a variety of assays, including flow cytometry, quantitative real-time PCR (qRT-PCR), or immunofluorescence assays.
Additionally, the methods outlined above are highly modular for essentially any gene circuit architecture expressing genes-of-interest, with the main potential protocol modifications&#160;at the circuit construction step and the assays conducted. In the case of gene circuit construction, the flexibility of molecular cloning allows any gene of interest to be exchanged or co-expressed with a fluorescence marker with minor modifications to primer design or assembly protocols. Additionally, while the procedures outlined here focus mostly on a NF gene circuit design for precise (low-noise) gene expression control, other architectures such as positive regulation or positive feedback (PF) can be implemented to achieve different features such as high-fold change or high gene expression noise, respectively56,57. Using a variety of gene circuit architectures (e.g., PF, NF, etc.) can allow researchers to explore diverse biological questions such as the roles protein level magnitudes and noise play in drug resistance or metastasis58. The protocols listed here also focus on various ways of gene expression quantification, but any number of functional assays (e.g., cell motility, wound-healing, proliferation, etc.) can be added after microscopy acquisition with little to no effect on the preceding methods. This is especially relevant to single-cell studies where optogenetics can use spatiotemporal induction to study behaviors such as pulsatile expression dynamics59.
Complementing method modularity, troubleshooting is another important feature of each main protocol. For circuit construction, the main differences resides in the appropriate circuit-specific primer design, while later methods such as assembly and plasmid amplification via bacterial growth are more standardized and typically include their own extensive troubleshooting procedures. Cell engineering can be modified with drug titration curves depending on the cell line used. Additionally, if a commercial cell line is used, troubleshooting guides can be obtained directly from the cell line manufacturer. Also, unintended light effects are important to quantify on the engineered cell lines. For example, due to the phototoxic effects of 470 nm light, a light intensity curve should be created to investigate the induction of death or damage in a population. Once a variety of light values are tested, users can create an FSC-SSC gate or utilize a method such as propidium iodide staining to quantify dead/damaged cells, and therefore, serve as a tool to determine appropriate light ranges to use experimentally.
For LPA troubleshooting, LEDs can produce edge effects of light and heat crosstalk with adjacent wells. For example, high-intensity wells may cause some induction in adjacent wells if there is an improper seal or a large heat/light gradient between wells. These effects can be maximal&#160;if the LPA is programmed in a particular order (e.g., ascending/descending) of a light parameter. To combat this, the IRIS software allows users to employ a randomization matrix to randomize the well intensities and therefore reduce systematic error. For troubleshooting aspects of fluorescence microscopy, exposure time, gain settings (controlling camera sensitivity), and light source intensity are the main parameters that can be adjusted. Tuning these parameters can allow for optimal image production and ideal signal-to-noise ratios as well as avoiding oversaturation and phototoxicity.
For flow cytometry troubleshooting, photomultiplier tube voltages and cellular gating are the main aspects that can be adjusted. Tuning these parameters can allow ideal comparisons between experimental conditions and control samples, proper signal acquisition for both weak or strong fluorescence signals, and exclusion of cellular debris or unwanted cell populations. It should be noted that flow cytometry voltages are ideally kept constant across experiments to allow proper comparisons. Additionally, for optogenetic systems requiring multiple fluorescence outputs (e.g., FITC and PE), users will need to be aware of fluorescence compensation given crosstalk between fluorophores. In such scenarios, controls are crucial for determining proper fluorescence signals. Controls that will be necessary for users to perform include fluorescent beads, stained cells with the marker of interest, or cells constitutively expressing the fluorescence protein that can be used for creating a compensation matrix in the flow cytometry software of interest. Additionally, whether using one fluorescence marker or more, all users should routinely measure fluorescence signals of each marker for non-fluorescent cells, which yield autofluorescence/background signals. qRT-PCR troubleshooting includes varying cDNA amounts and probe design, which are often project-specific. Lastly, for immunofluorescence troubleshooting, antibody concentrations are the biggest variable for assay quantification, necessitating optimal concentration determination.
One troubleshooting aspect that is relevant to most of the above methods is the isolation effect of using a sealed plate with cells covered with foil. This method may impede the carbon dioxide gas exchange needed for the proper growth of various cell lines. From previous experimental experience, this was not a significant impediment to cellular growth for a 3-5 day experiment using the engineered cell lines within this manuscript but may become important for other cell lines or experimental durations. To address this concern, one adaptation implemented with the sealed plates includes using carbon dioxide independent media, which has not affected the growth of cells used in this study. It should be noted for other assays or cell lines where metabolism, pH levels, and cell densities are important, other interventions may be needed such as changing media or nutrients more frequently.
The limits of the methods outlined here reside in the gene circuit used, the optogenetic technology precision, and LPA interface with other equipment such as fluorescence microscopes. The technology described here is affordable, quick to build, and easy to validate46 for populations of optogenetically engineered cells. However, there are certain spatial limitations, such as the inability to control single cells without modifications to the light induction setup, such as utilizing digital mirror devices (DMDs), with recently demonstrated benefits in living cells60,61. Furthermore, the methods outlined here are limited in live-cell tracking during optogenetic stimulation, allowing only for the end-point data acquisition of the entire experimental time course.
Despite these limits, the optogenetic methods outlined here are complementary to the existing technology such as the DMDs, which have the flexibility to control single cells in real-time but are limited by the number of samples one can stimulate. Furthermore, the stable cell line engineering methods discussed here contrast existing synthetic biology methods which often characterize engineered genetic systems via transient transfections. Transient transfection approaches are inherently noisier due to the greater gene circuit copy number variation among cells. In contrast, the methods presented here involve monoclonal cell variants, each containing one gene circuit copy. Stable cell lines allow exploring cellular aspects such as gene expression variation, protein level effects on phenotypic landscapes, and single-cell methods with finer precision since there is higher confidence that each cell is identical to its neighbors.
The work here demonstrates a platform for designing any genomically integrated optogenetic gene circuit of interest, inducing such systems with reliable experimental tools, and characterizing them with a variety of gene expression and functional/phenotypic assays in a standardized manner. Future improvements of these protocols may include the integration of these methods with live-cell tracking using other optogenetic technologies such as the DMDs, which will allow spatiotemporal control of gene expression and functional applications at the single-cell level. Such advances will allow light utilization to produce the gene expression pattern of interest in single cells, establish transcription/translation dynamics, quantify protein levels, and study their role in diverse biological processes, including cell migration, proliferation, metastasis, and differentiation.

Similar to other engineering disciplines, synthetic biology aims to standardize protocols, allowing tools with highly reproducible functions to be utilized for exploring questions relevant to biological systems1,2. One domain in synthetic biology where many control systems have been built is the area of gene expression regulation3,4. Gene expression control can target both protein levels and variability (noise or coefficient of variation, CV = &#963;/&#181;, measured as the standard deviation over the mean), which are crucial cellular characteristics due to their roles in physiological and pathological cellular states5,6,7,8. Many synthetic systems that can control protein levels and noise4,9,10,11,12 have been engineered, creating opportunities to standardize protocols across tools.
One novel set of tools that can control gene networks that has recently emerged is optogenetics, enabling the use of light to control gene expression13,14,15,16,17. Similar to their chemical predecessors, optogenetic gene circuits can be introduced into any cell type, ranging from bacteria to mammals, allowing expression of any downstream gene of interest18,19. However, due to the rapid generation of novel optogenetic tools, many systems have emerged that vary in genetic circuit architecture, mechanism of expression (e.g., plasmid-based vs. viral integration), and light supplying control equipment11,16,20,21,22,23,24,25. Therefore, this leaves room for standardization of optogenetic features such as gene circuit construction and optimization, method of system utilization (e.g., integration vs. transient expression), experimental tools used for induction, and analysis of results.
To make progress on standardizing optogenetic protocols in mammalian cells, this protocol describes an experimental pipeline to engineer optogenetic systems in mammalian cells using a negative feedback (NF) gene circuit integrated into HEK293 cells (human embryonic kidney cell line) as an example. NF is an ideal system to demonstrate standardization since it is highly abundant26,27,28 in nature, allowing protein levels to be tuned and noise minimization to occur. In brief, NF allows for precise gene expression control by a repressor reducing its own expression sufficiently fast, thereby limiting any change away from a steady state. The steady state can be altered by an inducer that inactivates or eliminates the repressor to allow for more protein production until a new steady state is reached for each inducer concentration. Recently, an engineered NF optogenetic system was created that can produce a wide-dynamic response of gene expression, maintain low noise, and respond to light stimuli allowing the potential for spatial gene expression control11. These tools, known as light-inducible tuners (LITers), were inspired by earlier systems that allowed gene expression control in living cells4,10,29,30 and were stably integrated into human cell lines to ensure long-term gene expression control.
Here, using the LITer as an example, a protocol is outlined for creating light-responsive gene circuits, inducing gene expression with a Light Plate Apparatus (LPA, an optogenetic induction hardware)31, and analyze responses of the engineered, optogenetically-controllable cell lines to custom light stimuli. This protocol allows users to utilize the LITer tools for any functional gene they wish to explore. It can also be adapted for other optogenetic systems with diverse circuit architectures (e.g., positive feedback, negative regulation, etc.) via integrating the methods and optogenetic equipment outlined below. Similar to other synthetic biology protocols, the video recordings and optogenetic protocols outlined here can be applied in single-cell studies in diverse areas, including but not limited to cancer biology, embryonic development, and tissue differentiation.

<table><tbody><tr><td>0.2 mL PCR tubes</td><td>Eppendorf</td><td>951010006</td><td>reagent for carrying out PCR</td></tr><tr><td>0.25% Trypsin EDTA 1X</td><td>Thermo Fisher Scientific</td><td>MT25053CI</td><td>reagent for splitting &amp;amp; harvesting mammalian cells</td></tr><tr><td>0.5-10 &amp;mu;L Adjustable Volume Pipette</td><td>Eppendorf</td><td>3123000020</td><td>tool used for pipetting reactions</td></tr><tr><td>100-1000 &amp;mu;L Adjustable Volume Pipette</td><td>Eppendorf</td><td>3123000039</td><td>tool used for pipetting reactions</td></tr><tr><td>20-200 &amp;mu;L Adjustable Volume Pipette</td><td>Eppendorf</td><td>3123000055</td><td>tool used for pipetting reactions</td></tr><tr><td>2-20 &amp;mu;L Adjustable Volume Pipette</td><td>Eppendorf</td><td>3123000039</td><td>tool used for pipetting reactions</td></tr><tr><td>5 mL Polystyene Round-Bottom Tube w/ Cell Strainer Cap</td><td>Corning</td><td>352235</td><td>reagent for flow cytometry</td></tr><tr><td>5702R Centrifuge, with 4 x 100 Rotor, 15 and 50 mL Adapters, 120 V</td><td>Eppendorf</td><td>22628113</td><td>equipment for mammalian culture work</td></tr><tr><td>Agarose</td><td>Denville Scientific</td><td>GR140-500</td><td>reagent for gel electrophoresis</td></tr><tr><td>Aluminum Foils for 96-well Plates</td><td>VWR&amp;reg;</td><td>60941-126</td><td>tool used for covering plates in light-induction experiments</td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>A9518-5G</td><td>reagent for selecting bacteria with correct plasmid</td></tr><tr><td>Analog vortex mixer</td><td>Thermo Fisher Scientific</td><td>02215365PR</td><td>tool for carrying PCR, transformation, or gel extraction reactions</td></tr><tr><td>Bacto Dehydrated Agar</td><td>Fisher Scientific</td><td>DF0140010</td><td>reagent for growing bacteria</td></tr><tr><td>BD LSRAria</td><td>BD</td><td>656700</td><td>tool for sorting engineered cell lines into monoclonal populations</td></tr><tr><td>BD LSRFortessa</td><td>BD</td><td>649225</td><td>tool for characterizing engineered cell lines</td></tr><tr><td>BSA, Bovine Serum Albumine</td><td>Government Scientific Source</td><td>SIGA4919-1G</td><td>reagent for IF incubation buffer</td></tr><tr><td>Cell Culture Plate 12-well, Clear, flat-bottom w/lid, polystyrene, non-pyrogenic, standard-TC</td><td>Corning</td><td>353043</td><td>plate used for growing monoclonal cells</td></tr><tr><td>Centrifuge</td><td>VWR</td><td>22628113</td><td>instrument for mammalian cell culture</td></tr><tr><td>Chemical fume hood</td><td>N/A</td><td>N/A</td><td>instrument for carrying out IF reactions</td></tr><tr><td>Clear Cell Culture Plate 24 well flat-bottom w/ lid</td><td>BD</td><td>353047</td><td>plate used for growing monoclonal cells</td></tr><tr><td>CytoOne T25 filter cap TC flask</td><td>USA Scientific</td><td>CC7682-4825</td><td>container for growing mammalian cells</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fischer Scientific</td><td>BP231-100</td><td>reagent used for freezing down engineered mammalian cells</td></tr><tr><td>Ethidium Bromide</td><td>Thermo Fisher Scientific</td><td>15-585-011</td><td>reagent for gel electrophoresis</td></tr><tr><td>Falcon 96 Well Clear Flat Bottom TC-Treated Culture Microplate, with Lid</td><td>Corning</td><td>353072</td><td>container for growing sorted monoclonal cells</td></tr><tr><td>FCS Express</td><td>De Novo Software:</td><td>N/A</td><td>software for characterizing flow cytometry data</td></tr><tr><td>Fetal Bovine Serum, Regular, USDA 500 mL</td><td>Corning</td><td>35-010-CV</td><td>reagent for growing mammalian cells</td></tr><tr><td>Fisherbrand Petri Dishes with Clear Lid - Raised ridge; 100 x 15 mm</td><td>Fisher Scientific</td><td>FB0875712</td><td>equipment for growing bacteria</td></tr><tr><td>Gibco&amp;nbsp;DMEM, High Glucose</td><td>Thermo Fisher Scientific</td><td>11-965-092</td><td>reagent for growing mammalian cells</td></tr><tr><td>Hs00932330_m1 KRAS isoform a Taqman Gene Expression Assay</td><td>Life Technologies</td><td>4331182</td><td>qPCR Probe</td></tr><tr><td>Hygromycin B (50 mg/mL), 20 mL</td><td>Life Technologies</td><td>10687-010</td><td>reagent for selecting cells with proper gene circuit integration</td></tr><tr><td>iScript Reverse Transcription Supermix</td><td>Bio-Rad Laboratories</td><td>1708890</td><td>reagent for converting RNA to cDNA</td></tr><tr><td>Laboratory Freezer -20 &amp;deg;C</td><td>VWR</td><td>76210-392</td><td>equipment for storing experimental reagents</td></tr><tr><td>Laboratory Freezer -80 &amp;deg;C</td><td>Panasonic</td><td>MDF-U74VC</td><td>equipment for storing experimental reagents</td></tr><tr><td>Laboratory Refrigerator +4 &amp;deg;C</td><td>VWR</td><td>76359-220</td><td>equipment for storing experimental reagents</td></tr><tr><td>LB Broth (Lennox) , 1 kg</td><td>Sigma-Aldrich</td><td>L3022-250G</td><td>reagent for growing bacteria</td></tr><tr><td>LIPOFECTAMINE 3000</td><td>Life Technologies</td><td>L3000008</td><td>reagemt for transfecting gene circuits into mammalian cells</td></tr><tr><td>MATLAB 2019</td><td>MathWorks</td><td>N/A</td><td>software for analyzing experimental data</td></tr><tr><td>Methanol</td><td>Acros Organics</td><td>413775000</td><td>reagent for immunofluorescence reaction</td></tr><tr><td>Microcentrifuge Tubes, Polypropylene 1.7 mL</td><td>VWR</td><td>20170-333</td><td>plasticware container</td></tr><tr><td>Mr04097229_mr EGFP/YFP Taqman Gene Expression Assay</td><td>Life Technologies</td><td>4331182</td><td>qPCR Probe</td></tr><tr><td>MultiTherm Shaker</td><td>Benchmark Scientific</td><td>H5000-HC</td><td>equipment for bacterial transformation</td></tr><tr><td>NanoDrop Lite Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-NDL-US-CAN</td><td>equipment for DNA/RNA concentration measurement</td></tr><tr><td>NEB Q5 High-Fidelity DNA polymerase 2x Master Mix</td><td>NEB</td><td>M0492S</td><td>reagent for PCR of gene circuit fragments</td></tr><tr><td>NEB10-beta Competent E. coli (High Efficiency)</td><td>New England Biolabs (NEB)</td><td>C3019H</td><td>bacterial cells for amplifying gene circuit of interest</td></tr><tr><td>NEBuilder HiFi DNA Assembly Master Mix</td><td>New England Biolabs (NEB)</td><td>E2621L</td><td>reagent for combining gene circuit fragements</td></tr><tr><td>Nikon Eclipse Ti-E inverted microscope with a DS-Qi2 camera</td><td>Nikon Instruments Inc.</td><td>N/A</td><td>instrument for quantifying gene expression</td></tr><tr><td>NIS-Elements</td><td>Nikon Instruments Inc.</td><td>N/A</td><td>software for characterizing fluorescence microscopy data</td></tr><tr><td>oligonucleotides</td><td>IDT</td><td>N/A</td><td>reagent used for PCR of gene circuit components</td></tr><tr><td>Panasonic MCO-170 AICUVHL-PA cellIQ Series CO&lt;sub&gt;2&lt;/sub&gt; Incubator with UV and H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; Control</td><td>Panasonic</td><td>MCO-170AICUVHL-PA</td><td>instrument for growing mammalian cells</td></tr><tr><td>Paraformaldehyde, 16% Electron Microscopy Grade</td><td>Electron Microscopy Sciences</td><td>15710-S</td><td>reagent</td></tr><tr><td>PBS, Dulbecco&#039;s Phosphate-Buffered Saline (D-PBS) (1x)</td><td>Invitrogen</td><td>14190144</td><td>reagent for mammalian cell culture,reagent for IF incubation buffer</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL), 100x</td><td>Fisher Scientific</td><td>15140-122</td><td>reagent for growing mammalian cells</td></tr><tr><td>primary ERK antibody</td><td>Cell Signaling Technology</td><td>4370S</td><td>primary ERK antibody for immunifluorescence</td></tr><tr><td>primary KRAS antibody</td><td>Sigma-Aldrich</td><td>WH0003845M1</td><td>primary KRAS antibody for immunifluorescence</td></tr><tr><td>QIAprep Spin Miniprep Kit (250)</td><td>Qiagen</td><td>27106</td><td>reagent kit for purifying gene circuit plasmids</td></tr><tr><td>QIAquick Gel Extraction Kit (50)</td><td>Qiagen</td><td>28704</td><td>reagent kit for purifying gene circuit fragments</td></tr><tr><td>QuantStudio 3 Real-Time PCR System</td><td>Eppendorf</td><td>A28137</td><td>equipment for qRT-PCR</td></tr><tr><td>Relative Quantification App</td><td>Thermo Fisher Scientific</td><td>N/A</td><td>software for quantifying RNA/cDNA amplificaiton</td></tr><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>74134</td><td>kit for extracting RNA of engineered mammalian cells</td></tr><tr><td>Secondary ERK antibody</td><td>Cell Signaling Technology</td><td>8889S</td><td>secondary ERK antibody for immunifluorescence</td></tr><tr><td>secondary KRAS antibody</td><td>Invitrogen</td><td>A11005</td><td>secondary KRAS antibody for immunifluorescence</td></tr><tr><td>Serological Pipets 5.0 mL</td><td>Olympus Plastics</td><td>12-102</td><td>reagents used for setting up a variety of chemical reactions</td></tr><tr><td>SmartView Pro Imager System</td><td>Major Science</td><td>UVCI-1200</td><td>tool for imaging correct PCR bands</td></tr><tr><td>SnapGene Viewer (free) or SnapGene</td><td>SnapGene</td><td>N/A</td><td>software DNA sequence design and analysis</td></tr><tr><td>Stage top incubator</td><td>Tokai Hit</td><td>INU-TIZ</td><td>tool for carrying PCR, transformation, or gel extraction reactions</td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher Scientific</td><td>4444557</td><td>reagent for PCR of gene circuit fragments</td></tr><tr><td>TaqMan Human GAPD (GAPDH) Endogenous Control (VIC/MGB probe), primer limited, 2500 rxn</td><td>Life Technologies</td><td>4326317E</td><td>qPCR Probe</td></tr><tr><td>Thermocycler</td><td>Bio-Rad</td><td>1851148</td><td>tool for carrying PCR, transformation, or gel extraction reactions</td></tr><tr><td>VisiPlate-24 Black, Black 24-well Microplate with Clear Bottom, Sterile and Tissue Culture Treated</td><td>PerkinElmer</td><td>1450-605</td><td>plate used for light-induction experiments</td></tr><tr><td>VWR Disposable Pasteur Pipets, Glass, Borosilicate Glass Pipet, Short Tip, Capacity=2 mL, Overall Length=14.6 cm</td><td>VWR</td><td>14673-010</td><td>reagent for mammalian cell culture</td></tr><tr><td>VWR Mini Horizontal Electrophoresis Systems, Mini10 Gel System</td><td>VWR</td><td>89032-290</td><td>equipment for DNA gel electrophoresis</td></tr><tr><td>Flp-In 293</td><td>Thermo Fisher Scientific</td><td>R75007</td><td>Engineered cell line with FRT site</td></tr></tbody></table>

reliably engineering and controlling stable optogenetic gene circuits in mammalian cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Watch this Scientific Journal Video about Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells at JoVE.com

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

reliably-engineering-controlling-stable-optogenetic-gene-circuits

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.3K Views.  Stony Brook University. Het gestandaardiseerde optogenetische protocol is ontworpen voor laboratoria die geïnteresseerd zijn in het gebruik van licht als de stimulus om cellulaire eigenschappen in gemanipuleerde zoogdiercelsystemen te regelen. Dit protocol is eenvoudig te implementeren in laboratoria die anders niet bekend zijn met optogenetica en biedt methoden voor nauwkeurige ruimtelijke temporele controle van gemanipuleerde biologische systemen. Deze methode heeft een breed scala aan toepassingen in specifieke ...

Video: Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetics

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Developmental Biology

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Neuroscience

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Behavior

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

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