Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Summary

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.

Abstract

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.

Introduction

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 systems^1,2. One domain in synthetic biology where many control systems have been built is the area of gene expression regulation^3,4. Gene expression control can target both protein levels and variability (noise or coefficient of variation, CV = σ/µ, measured as the standard deviation over the mean), which are crucial cellular characteristics due to their roles in physiological and pathological cellular states^5,6,7,8. Many synthetic systems that can control protein levels and noise^4,9,10,11,12 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 expression^{13,14,15,16,17}. 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 interest^18,19. 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 equipment^{11,16,20,21,22,23,24,25}. 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 abundant^26,27,28 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 cells^4,10,29,30 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.

Protocol

1. Gene circuit design

1. Select genetic components to combine into a single gene circuit/plasmid (e.g., mammalian DNA integration sequence motifs³², light-responsive elements³³, or functional genes³⁴).
2. Using any genetic engineering and/or molecular cloning software, store the DNA sequences for later use and reference, annotate each sequence, and examine all the necessary features (e.g., START codons, regulatory, or translated sequences)³⁵.
3. Develop or adopt parts according to the overall gene circuit design³⁶. As an example, for optogenetic repressors as in the LITers¹¹, fuse a gene sequence coding for a domain capable of light-inducible degradation^37,38 or inactivation of a repressor's DNA binding ability³⁹, adjacent to and in the frame of the repressor gene (e.g., TetR)⁴.
   1. For optogenetic activators, ensure the presence of an activator domain⁴⁰ that promotes gene expression upon light-induced DNA binding⁴¹. For negative or positive autoregulation, ensure the presence of a regulatory binding site in or upstream of the regulator gene's promoter (e.g., tetO sites in or upstream of the TetR expressing promoter)⁴².
4. Design the primers for DNA sequence amplification or sequencing of the plasmid using the molecular cloning software for each gene circuit.
5. Validate the primers computationally for plasmid construction through the built-in feastures of molecular cloning software (e.g., sequence alignment).
6. Order oligonucleotide primers from the manufacturer. Plasmids constructed and used in this work can be found in the original supporting material¹¹ along with the primers designed and used.
7. Dilute the primers to 100 mM stock concentration in double-distilled water (ddH₂O).
8. Dilute the stock of 100 mM stock primers to 10 mM concentration for PCR.
9. Prepare the PCR mix with 1 µL of forward primer, 1 µL of reverse primer, 1 µL of template DNA to a total mass of 0.5-500 ng for genomic or 0.5 pg-5 ng for plasmid or viral DNA, 12.5 µL of DNA polymerase 2x master mix (or the volume that satisfies the manufacturer's dilution factor), and 9.5 µL of ddH₂O for a total reaction volume of 25 µL.
10. Incubate the PCR mix in a thermocycler at appropriate settings depending on the enzyme chosen⁴³. Suggested reaction cycles include:
    Step 1: One cycle of 30 s initial denaturation step at 95 °C.
    Step 2: 40 cycles of 5 s of denaturation step at 98 °C.
    Step 3: One cycle of 30 s of annealing step at 65 °C (determined by primers designed earlier).
    Step 4: One cycle of 1 min of extension at 72 °C (~1 kilobase (kb) template fragment length).
    Step 5: One cycle of a 2 min extension at 72 °C.
    Hold the reaction at 4 °C until it can be tested via gel electrophoresis. This protocol will vary depending on the reagents used (e.g., polymerase and buffers).
11. Repeat step 1.9 to amplify all the fragments to be used in a DNA assembly reaction required for linking linear PCR products into a single circular DNA vector.
12. Run the PCR products on a 1% agarose gel followed by purifying the bands of the desired length.
13. Prepare the master mix for a DNA assembly reaction (Table 1).
    NOTE: In this example, the mother-vector is split into two fragments to increase the efficiency of the PCR steps without causing significant changes to the overall assembly outcome.
14. Incubate the DNA assembly reaction master mix in a thermocycler at 50 °C for 1 h (unless otherwise specified in DNA assembly reagent protocol) and store the reaction at 4 °C to use the reaction product for bacterial transformation.
15. Set up bacterial transformation (chemical or electroporation) using competent E. coli (Escherichia coli) cells and the corresponding bacterial transformation protocol. After transformation, use the bacterial Luria-Bertani (LB) agar plates containing the chosen bacterial selection marker (e.g., Ampicillin) to plate the transformation mix. Incubate the plates at 37 °C overnight⁴⁴.
16. Check the plates the following day. To inoculate the colonies, pick individual colonies from the plates and resuspend them in a liquid LB broth with the corresponding bacterial selection marker in culture tubes. Incubate in a shaker incubator at 37 °C, 300 rpm overnight.
17. Perform plasmid preparation protocol to extract the plasmid DNA from the bacterial culture.
18. Validate the circuit in two steps. First, perform a test digestion using restriction enzymes as a crude verification to see whether the approximate plasmid product was obtained. Second, if the test digestion is passed/confirmed, submit the plasmid to a Sanger sequencing facility (or process using the available equipment) to obtain the precise DNA sequence to compare it later to the expected sequence in the design software.
    1. To perform test digestion, select at least two restriction enzymes that produce at least two fragments, based on the molecular cloning software used. Once enzymes are selected, prepare the test samples by adding 1 µL of each enzyme, 5 µL of the generated DNA, and 13 µL of water with appropriate salts and buffers depending on the enzymes used. Incubate the reaction at 37 °C for 1 h, or as the enzyme manufacturer suggests. Run the test digestion products on a 1% agarose gel and determine whether the bands are correct.
       NOTE: If the bands are correct, proceed to sequencing.
    2. To perform sequencing, generate primers based on the DNA stored in the software so that the annealing regions of the primers are about 500 bp (base pairs) apart and cover the fragment of interest (gene circuit component) or the full plasmid. Dilute the primers with pure nuclease-free water (NF-H₂O) to 10 mM concentration. Prepare the sequencing samples by adding 1 µL of 10 ng/µL DNA, 1 µL of primer, and 8 µL of NF-H₂O to a 0.2 mL tube. Repeat this for each primer.
       NOTE: When samples are prepared, drop them off at the sequencing facility and compare the results with plasmid sequence using molecular cloning software.
19. At this step, generate approximately 100-1000 ng/µL of DNA sample for integrating the plasmids containing gene circuits into the appropriate cell line.

2. Stable cell line engineering

1. Order a mammalian cell line designed for rapid generation of stable sub-lines that ensure high-level expression of the protein of interest from a mammalian expression vector. The cell type and the ease of cell line engineering can be variable depending on what the users prefer or aim to achieve.
   1. For example, if users prefer cell line engineering with minimal intermediate steps, order the cells that contain a single stable integration site (e.g., FRT) at a transcriptionally active genomic locus. If more nuanced cell engineering is preferred, create integration sites at preferred locations using genetic engineering tools such as CRISPR/Cas9.
2. Grow cells in 5% CO₂ in humidified air at 37 °C. Adjust growth conditions as needed for the cell type.
3. Transfect the gene circuits designed above in the desired cells obtained from the previous steps to begin a stable cell-line generation process. To achieve this, use a liposome mixture⁴⁵ of the gene circuit DNA with appropriate recombinase (for example, Flp-recombinase for Flp-FRT recombination) or through other methods such as electroporation.
4. Two days after transfection, split the cells to 25% confluency.
5. Six hours after splitting the cells, begin antibiotic selection by exchanging the media to a fresh media containing 50 µg/mL of hygromycin antibiotic (or another antibiotic agent corresponding to the mammalian antibiotic resistance gene chosen during plasmid construction).
   NOTE: There are a variety of mammalian antibiotic resistance genes utilized in gene circuit construction, each of which has a different kill curve. Therefore, whichever mammalian antibiotic resistance gene is chosen for circuit construction, a proper kill curve should be studied in the cells of interest. This step ensures that cells containing the gene circuit payload are enriched while those without the system are killed off.
6. Allow the cells to grow in the antibiotic selection media, change to fresh media every 2-3 days until the plate or flask has a few dozen foci. Passage adherent cultures when they are in the log phase before they reach confluence.
7. Once there are sufficiently many foci, trypsinize the cells with 1 mL of 0.25% trypsin, 0.1% EDTA in Hank's balanced salt solution (HBSS) without Calcium, Magnesium, and Sodium Bicarbonate for several minutes. Neutralize the trypsin with fresh media and pass all the cells into a fresh container.
8. Once the cells in the fresh container are 80%-100% confluent, freeze them down in a mix of 45% old media, 45% fresh media, and 10% DMSO. Transfer the remaining cells to a sterile tube and perform single-cell sorting to isolate monoclonal cells into a 96-well plate.
9. Approximately 2-3 weeks post monoclonal sorting, wells within the 96-well plate should have foci. When approximately 50%-60% confluent, split the cells into a 12-well plate.
10. Once the 12-well plate is 80%-100% confluent, split into a tissue culture treated T-25 flask. Once the cells in the T-25 flask are 80%-100% confluent, freeze the cells and maintain a passage for characterization and testing of monoclonal cell lines.
    1. Characterize the monoclonal cell lines by microscopy and flow cytometry assays to report gene expression profiles based on the induced fluorescent reporter production. Verify functional protein production via fluorescent antibody labeling and immunofluorescence assay. Test the transcriptional-level gene expression induction via quantitative real-time PCR (qRT-PCR). Validate the genetic sequence and integration accuracy via local and whole-genome sequencing of the clones.

3. Light plate apparatus induction assays

1. Construct an LPA device^31,46 to be used for light induction of engineered cells. Briefly, several broad steps are crucial for creating LPA to use for controlling gene expression. These include 3D printing LPA frame components, circuit board construction, programming the circuit via microcontroller programmer, assembling the components into a final LPA, programming the memory card via IRIS software, and calibrating the finished device. For a more thorough explanation, refer to the above references.
   1. Print the 3D parts as outlined in Gerhardt et al. (2016 and 2019)^31,46.
   2. Assemble the circuit board as outlined in Gerhardt et al. (2016 and 2019)^31,46.
   3. Add the firmware to the assembled LPA circuit using microcontroller programmer as outlined in Gerhardt et al. (2016 and 2019)^31,46.
   4. Combine 3D printed parts and the assembled circuit board as outlined in Gerhardt et al. (2016 and 2019)^31,46. This includes taking the mounting plate, the circuit board, the LED (light-emitting diodes) spacer, the plate adaptor, a 24-well plate, a plate lid, mounting bolts, and wing nuts and stacking components as shown in the filmed video and Figure 2.
   5. For memory card programming and calibration of the device, follow the steps described below.
2. Use the IRIS software available on the Tabor Lab website⁴⁷ to program an SD card for the Light Plate Apparatus (LPA)³¹ and explore appropriate light conditions needed to begin an optogenetic experiment.
   NOTE: The IRIS software is a web-based application for programming the optogenetic electronic hardware, known as the LPA, developed by the Tabor Lab. The software allows programming of relative IRIS values that control the individual light-emitting diodes (LEDs) in each well of the LPA hardware.
3. Choose the LPA (4 x 6) drop-down option, followed by clicking the appropriate illumination approach (Steady-state, Dynamic, Advanced).
   NOTE: For this manuscript, all the assays will focus on the steady-state examples. However, the advanced setting examples can be used for pulse durations and duty cycle regimes.
4. Choose whether the top or the bottom LEDs will be illuminated by entering values in the cells corresponding to the wells of the plate. For the LPAs used in this work, blue LEDs placed in the top position were used in all experiments. Each LED, once programmed, can provide continuous light exposure with a constant light intensity. The IRIS software allows for 4096 intensity levels which can be programmed.
5. Once the LED locations are chosen, enter intensity values for the desired experimental outline. For example, enter 8 different light intensities (or pulse durations or duty cycles) with three technical replicates per plate (Table 2). G.s. refers to grayscale units-the light intensity level measurement values used for LPA programming in the IRIS software.
   1. When designing IRIS experimental files, keep in mind edge effects from adjacent wells on the LPA device, light toxicity from increasing intensities of blue light exposure, and determining the type of dose-response desired (e.g., monotonic vs. non-monotonic).
   2. If users program cells in the LPA in ascending/descending order of a particular light parameter (e.g., intensity), wells may produce edge effects of light crosstalk or even heat which can influence adjacent wells. This can inadvertently influence the outcome of measured outputs when experiments are complete. To alleviate this, users can implement a randomization matrix on the IRIS software to scramble well locations, minimizing edge effects. An example is described in the Representative Results below (Figure 4A-B).
   3. In addition, higher intensities of blue light have been found to interfere with cellular growth and viability⁴⁸. Therefore, to mitigate light toxicity, it is important to produce a light-intensity, pulse duration, or duty cycle response curve depending on the modality being investigated.
      1. For example, program 8 light intensity values with three replicates per 24-well plate, with a range from no light to a max intensity of the LPA. Then, run these samples on a flow cytometer with an SSC-FSC gate or a live stain such as propidium iodide to quantify the population cell survival (living cells compared to total events including dead cells/cellular debris) at each light value.
      2. Then, determine the ideal amount of population survival for the experimental setup, since any stimulus may adversely affect proliferation, gene expression, or survival (e.g., setting 80% cell survival as an appropriate tradeoff). For example, in this work, once calibrated, the intensity did not exceed 3000 g.s. units (~3/4 the maximum intensity of the LPA device). An example of this is described in the  Representative Results below.
   4. In addition to restricting light values because of toxicity, users may wish to restrict light values to characterize a particular part of the dose-response, such as the range of the monotone response.
      1. To achieve this, initially scan a wide range of light intensities, pulse durations or duty cycles depending on the modality being analyzed when determining the desired dose-response (Table 2) to narrow in on the light regime of interest, e.g., where gene expression correlates positively with increasing light values for a monotonic dose-response.
      2. To determine the light range of interest, program a single LPA with up to 24 wells of different intensity/pulse duration/duty cycle/etc. or more wells (e.g., 48, 72, 96, etc.) depending on whether multiple LPAs are calibrated to deliver equivalent light amounts and proceed with the cell culture work or assays outlined below. Therefore, start characterization of an optogenetic system with a wide dose range of light stimuli to determine the range interval that gives the desired gene expression and subsequently perform experiments in that refined dose range.
      3. For example, in this work, once 3000 g.s. units was determined as the threshold for toxic light intensity; this threshold was used as the upper bound of light for assays outlined below (e.g., immunofluorescence).
         NOTE: The steps above are independent of the optical calibration of the LPA and refer to a molecular-level calibration for each optogenetic system.
6. Once the appropriate light intensity values are programmed in IRIS, insert a memory card with the USB 3.0 outlet into the LPA to download and transfer the files.
7. If calibration of the LPA is complete³¹, proceed to cell culture work for initiation of light-induction assay. If the calibration has not been done, calibrate the LPA using an Image analysis method (steps 3.7.1-3.7.3) once the LPA is programmed with the microcontroller programmer.
   1. Briefly program the LPA to have the same IRIS level as described by Gerhardt et al. (2016)³¹.
      NOTE: For calibration, the LPA was programmed to have a value of 2000 g.s.
   2. Once the device is programmed with the memory card and the reset button (physical button on the LPA) is pressed, acquire an image of the entire device (e.g., gel station imager, scanner device, etc.). For the calibration, acquire two images with the device rotated by 180°.
   3. Then, use open-source software designed by Gerhardt et al. (2016)³¹ to show the variability in LED intensity on the LPA plate and calculate the LED compensation values to make the intensity equal across wells. An example of this is described in the represented results below (Figure 3). Once this calibration is complete, proceed to cell culture work for initiation of light-induction assay.
8. Obtain flasks of engineered monoclonal cells from the previous section to investigate light-induction effects on gene expression.
9. Remove the old media from the flask.
10. Add 1 mL of trypsin to the cells and incubate for 5 min.
11. After 5 min, neutralize trypsin by adding 4 mL of Dulbecco-modified Eagle's medium (DMEM or other desired media) supplemented with the necessary chemicals (this work used 50 µg/mL of hygromycin, 1% penicillin/streptomycin solution, 10% fetal bovine serum, FBS).
12. Add ~75,000 cells per well in a 24-well black plate in a total volume of 500 µL. The number of cells seeded can vary depending on the duration of the experiment desired and the cell type used.
    NOTE: Additionally, note that cell seeding density affects the culture maintenance and potentially the duration of the experiment. Starting at a lower number of cells per well ensures longer time durations before the culture reaches confluency. Furthermore, the type of optogenetic components integrated into the gene circuits of interest will influence when gene expression reaches a steady state and therefore affect the duration of the experiments. Other factors that may be considered are cell line-specific growth rates, media composition, and conditional growth effects (i.e., light).
13. After plating, place the cells in a humidified incubator with 5% CO₂ to allow them to settle for 2-6 h.
14. After the incubation, transfer the cells to a tissue culture hood, remove the plastic lid, and add an adhesive foil strip to the top of the plate (Figure 2D). This step allows minimal light transfer between wells, as the top and the sides of the wells are coated, and the light can now only enter from the bottom of the well where the LED is placed.
15. Place the plate in the fitted 3D parts of the LPA. Then, cover the plate with the 3D-printed LPA lid, which fits over the device screws on each corner.
16. Plug the device into the power source and push the reset button on the LPA device to ensure that the updated LPA experiment settings are applied.
17. Incubate the cells within the experimental system for an appropriate amount of time, depending on the original seeding density, cell line-specific growth speed, and growth conditions. In this example, the cell lines were induced for 3 days continuously for gene expression to reach a steady state. However, it should be noted that many optogenetic systems (e.g., VVD) may reach steady-state gene expression much sooner (e.g., 24 h), and therefore experimental induction times can be reduced or prolonged as needed.
18. At the end of the light induction experiment, utilize the samples for any of the following four assays to characterize the engineered cell lines (sections 4-7).

4. Fluorescence microscopy of light-induced engineered cells

1. 24-72 h post-induction, remove the cells in the LPA from the incubator and place them in a tissue culture hood.
2. Remove the foil strip and let the plate sit for 1-2 min. This prevents condensation from forming on the plastic lid, if placed immediately on the plate.
3. After 1-2 min of sitting, put the original plastic lid back on the plate.
4. Image the cells with the appropriate phase contrast or fluorescence microscope.
5. Depending on the instrument, adjust the exposure time, light source intensity, and gain for displaying engineered cells. In this experiment, the following parameters were applied: 50 ms for FITC/GFP (green fluorescent protein) light source exposure time and 1-5 ms for phase-contrast exposure time at 100% intensity for each.
   NOTE: It should be noted that optimal exposure times, gain levels, and light source intensities are often derived empirically from the experience of this work to minimize oversaturation in the fluorescence reporter, minimize cellular damage, and capture adequate images for qualitative and quantitative analysis. When determining the levels of each of these parameters, the aspects to keep in mind include maximizing signal-to-noise ratios, minimizing phototoxicity, minimizing oversaturation of fluorescence signals, and increasing the ability to amplify weak fluorescence signals.
   1. Optimize these parameters grossly ad-hoc; however, previous experimental values (e.g., light source intensity causing oversaturation of fluorescence reporter) can guide future experimental settings when applicable. For example, the gain settings, exposure times, and light intensity initial values from one circuit (LITer1.0) or experimental setup (e.g., light intensity) can be used as a starting point when moving to a similar but different gene circuit (LITer2.0)¹¹ or a different light modality (e.g., light duty cycle).
6. Streamline plate imaging by a coordinate template programmed into the microscopy software allowing the entire plate size (e.g., 24 wells) to have automatized image acquisition from multiple image locations per well.

5. Flow cytometry of light-induced engineered cells

1. 24-72 h post-induction, remove the cells from the LPA in the incubator or from the microscope after imaging and place them in the tissue culture hood.
2. If removed directly from the LPA, remove the foil strip, and let the plate to sit for a minute or two.
3. Aspirate the media from each well (of the entire 24-well plate if all the wells are used).
4. Add 100 µL of 0.25% trypsin to each well, cover the plate with a plastic lid, and place it back in the incubator.
5. Leave the cells undisturbed in the incubator for 5 min.
6. After 5 min, remove the plate and return it to the tissue culture hood.
7. Neutralize each trypsinized well with 400 µL of DMEM.
8. Label a 5 mL polystyrene round-bottom tube with cell strainer (or appropriate flow cytometry tube that can be filtered to remove large clumps of cells not fully trypsinized) with a name corresponding to each well.
9. Use a P1000 pipette to pipette the contents of each well up and down 6-8 times to break cell clumps and create single-celled samples for flow cytometry⁴⁹.
10. Transfer the entire contents of each well (~500 µL) to the labeled tubes with the strainer.
11. Bring cells to the appropriate flow cytometry instrument with lasers of the correct wavelengths (can bring tubes with cells on or off ice).
    NOTE: The flow cytometer used in this work was a part of a core facility located at the university hospital.
12. Create a forward- and side-scatter gate (FSC and SSC, respectively) to capture the single cells of the appropriate size and granularity to exclude debris and cellular clumps on the flow cytometry software.
13. Once the gate is set, capture approximately 10,000 cells with the appropriate gate. Adjust this number depending on the amount of cellular data the users are seeking. Repeat for each tube containing the cells from the experiment.
14. Once the experiment is complete, import the data to the available flow cytometry data software for analysis.
15. Create an FSC-SSC gate (as before during acquisition) and apply it to each batch of experimental data. A reference well of the un-induced cell population is used for creating this gate in this manuscript, but other metrics for gate creation exist, such as density-based gates.
16. With the experimental conditions gated with an FSC-SSC gate, plot the flow cytometry data as histograms or represent in other ways to illustrate the obtained expression patterns. In this experiment, the fluorescence was captured by the GFP/FITC or PE/TexasRed channels.

6. RNA extraction and quantitative PCR of gene circuit components

1. 24-72 h post-induction, remove the cells from the LPA in the incubator or from the microscope after imaging and place them in the tissue culture hood.
2. If removed directly from the LPA, remove the foil strip, and let the plate sit for a minute or two.
3. Aspirate the media from each well (of the entire 24-well plate if all the wells are used).
4. Proceed to extract RNA from the cells using the appropriate RNA extraction kit.
5. Once the RNA extraction is complete, perform a reverse transcription reaction of each sample (Table 3).
6. Further, perform quantitative PCR of each sample (Table 4). Utilize a DNA polymerase and associated protocol to set up PCR reactions. For this step, set up a multiplexed reaction with a housekeeping gene and a gene of interest, or create separate reactions. In this example, GFP, KRAS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were probed. After completing the qRT-PCR experiment, proceed with the analysis via available software to illustrate gene circuit fold change at the RNA level.

7. Immunofluorescence of gene circuit components

1. Use an ice bath or freezer to cool down methanol.
2. 24-72 h post-induction, remove the cells from the LPA in the incubator or from the microscope after imaging and place them in the tissue culture hood.
3. If removed directly from the LPA, remove the foil strip, and let the plate sit for 1-2 min.
4. Aspirate the media from each well (of the entire 24-well plate if all the wells are used).
5. Add 100 µL of 0.25% trypsin to each well, cover the plate with a plastic lid, and place it back in the incubator.
6. Leave the cells undisturbed in the incubator for 5 min.
7. After 5 min, remove the plate and return it to the tissue culture hood.
8. Neutralize each trypsinized well with 400 µL of DMEM.
9. Label mini-centrifuge tubes with names corresponding to each well.
10. Use a P1000 pipette and pipette the contents of each well up and down 6-8 times to break the cell clumps.
11. Transfer the entire contents of each well (~500 µL) to the labeled tubes.
12. Centrifuge the cells for 5 min at 400 x g.
13. When complete, discard the supernatant and move the samples to a chemical fume hood.
14. Resuspend the cells (use a P1000 pipette and pipette up and down 6-8 times in each tube to break cell clumps) in 750-1000 µL of 4% paraformaldehyde (diluted in phosphate-buffered saline, PBS).
15. Allow the cells to sit for 15 min at room temperature.
16. After the incubation, add 750-1000 µL of PBS. Pipette up and down several times.
17. Centrifuge the cells for 5 min at 400 x g.
18. When complete, discard the supernatant and move the samples to a chemical fume hood.
19. Resuspend the cells (use a P1000 pipette and pipette up and down 6-8 times in each tube to break cell clumps) in 750-1000 µL of ice-cold methanol.
20. Allow the cells to sit for 30 min on ice or in a -20 °C freezer.
21. After the incubation, add 750-1000 µL of PBS. Pipette up and down several times.
22. Centrifuge the cells for 5 min at 400 x g.
23. When complete, discard the supernatant and move the samples to a chemical fume hood.
24. Depending on the type of antibodies used, modify the protocol from this point forward. Follow either steps 7.24.1-7.24.14 or 7.24.15-7.24.22.
    1. If using a primary and secondary antibody, resuspend the cells using a P1000/P200 pipette and pipette up and down 6-8 times in each tube to break cell clumps in 100 µL of primary antibody. In this case, a dilution of 1:800 for stock antibodies was made, including KRAS antibody or ERK antibody, and was allowed to sit for 1 h at room temperature. Antibodies were diluted in an incubation buffer made from 1x PBS with 0.5 g of BSA.
       NOTE: Determine the exact dilution of the antibody empirically by creating a standard curve of antibody dilutions vs. the antigen of interest.
    2. After adding antibodies, cover the tubes with a foil to prevent light effects on labeled antibodies.
    3. After incubation, add 750-1000 µL of the incubation buffer. Pipette up and down several times.
    4. Centrifuge the cells for 5 min at 400 x g.
    5. When complete, discard the supernatant and move the samples to a chemical fume hood.
    6. Resuspend the cells using a P1000/P200 pipette and pipette up and down 6-8 times in each tube to break cell clumps in 100 µL of secondary antibody. In this case, cells were resuspended in 100 µL secondary antibody at a dilution of 1:800 for KRAS antibody or 1:2000 for ERK antibody and allowed to sit for 30 min at room temperature. Similar to primary antibodies, dilute the secondary antibodies in the incubation buffer as described above. Determine the dilutions of the secondary antibodies based on the empirical findings of a standard curve.
    7. After adding antibodies, cover the tubes with a foil to prevent light effects on the labeled antibodies.
    8. After the incubation, add 750-1000 µL of the incubation buffer. Pipette up and down several times.
    9. Centrifuge the cells for 5 min at 400 x g.
    10. When complete, discard the supernatant and move the samples to a chemical fume hood.
    11. Resuspend the cells using a P1000 pipette and pipette up and down 6-8 times in each tube to break cell clumps in 500 µL of PBS.
    12. Transfer the entire contents of each tube (~500 µL) to the labeled tubes with strainers.
    13. Bring the cells to appropriate flow cytometry instruments with lasers of the correct wavelengths (can bring tubes with cells on or off ice).
        NOTE: It should be noted that having several controls are important for progressing with flow cytometry measurement and analysis of engineered cell gene expression. For example, having completely unstained cells, cells stained with primary antibody alone, and cells stained with secondary antibody alone can be useful for comparing results with background signals from antibodies.
    14. Next, proceed with the analysis as described in section 5.
    15. If using only a primary antibody, resuspend the cells using a P1000/P200 pipette and pipette up and down 6-8 times in each tube to break cell clumps in 100 µL of labeled primary antibody. Determine appropriate antibody dilution and incubate for 1 h at room temperature. Determine the antibody dilution from the standard curve empirically.
    16. After adding antibodies, cover the tubes with a foil to prevent light effects on labeled antibodies.
    17. After incubation, add 750-1000 µL of the incubation buffer. Pipette up and down several times.
    18. Centrifuge the cells for 5 min at 400 x g.
    19. Resuspend the cells using a P1000 pipette and pipette up and down 6-8 times in each tube to break cell clumps in 500 µL of PBS.
    20. Transfer the entire contents of each tube (~500 µL) to labeled tubes with strainers.
    21. Bring the cells to appropriate flow cytometry instruments with lasers of the correct wavelengths (can bring tubes with cells on or off ice).
        NOTE: It should be noted that having several controls are important for progressing with flow cytometry measurement and analysis of engineered cell gene expression. Having completely unstained cells and cells stained with primary antibody alone are useful for comparing results with background signals from antibodies.
    22. From this point, proceed with the analysis as described in section 5.

Results

Gene circuit assembly and stable cell line generation within this article were based on commercial, modified HEK-293 cells containing a transcriptionally active, single stable FRT site (Figure 1). The gene circuits were constructed into vectors that had FRT sites within the plasmid, allowing for the Flp-FRT integration into the HEK-293 cell genome. This approach is not limited to Flp-In cells, as FRT sites can be added to any cell line of interest anywhere in the genome using DNA editing tec...

Discussion

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 characterizat...

Materials

Name	Company	Catalog Number	Comments
0.2 mL PCR tubes	Eppendorf	951010006	reagent for carrying out PCR
0.25% Trypsin EDTA 1X	Thermo Fisher Scientific	MT25053CI	reagent for splitting & harvesting mammalian cells
0.5-10 μL Adjustable Volume Pipette	Eppendorf	3123000020	tool used for pipetting reactions
100-1000 μL Adjustable Volume Pipette	Eppendorf	3123000039	tool used for pipetting reactions
20-200 μL Adjustable Volume Pipette	Eppendorf	3123000055	tool used for pipetting reactions
2-20 μL Adjustable Volume Pipette	Eppendorf	3123000039	tool used for pipetting reactions
5 mL Polystyene Round-Bottom Tube w/ Cell Strainer Cap	Corning	352235	reagent for flow cytometry
5702R Centrifuge, with 4 x 100 Rotor, 15 and 50 mL Adapters, 120 V	Eppendorf	22628113	equipment for mammalian culture work
Agarose	Denville Scientific	GR140-500	reagent for gel electrophoresis
Aluminum Foils for 96-well Plates	VWR®	60941-126	tool used for covering plates in light-induction experiments
Ampicillin	Sigma Aldrich	A9518-5G	reagent for selecting bacteria with correct plasmid
Analog vortex mixer	Thermo Fisher Scientific	02215365PR	tool for carrying PCR, transformation, or gel extraction reactions
Bacto Dehydrated Agar	Fisher Scientific	DF0140010	reagent for growing bacteria
BD LSRAria	BD	656700	tool for sorting engineered cell lines into monoclonal populations
BD LSRFortessa	BD	649225	tool for characterizing engineered cell lines
BSA, Bovine Serum Albumine	Government Scientific Source	SIGA4919-1G	reagent for IF incubation buffer
Cell Culture Plate 12-well, Clear, flat-bottom w/lid, polystyrene, non-pyrogenic, standard-TC	Corning	353043	plate used for growing monoclonal cells
Centrifuge	VWR	22628113	instrument for mammalian cell culture
Chemical fume hood	N/A	N/A	instrument for carrying out IF reactions
Clear Cell Culture Plate 24 well flat-bottom w/ lid	BD	353047	plate used for growing monoclonal cells
CytoOne T25 filter cap TC flask	USA Scientific	CC7682-4825	container for growing mammalian cells
Dimethyl sulfoxide (DMSO)	Fischer Scientific	BP231-100	reagent used for freezing down engineered mammalian cells
Ethidium Bromide	Thermo Fisher Scientific	15-585-011	reagent for gel electrophoresis
Falcon 96 Well Clear Flat Bottom TC-Treated Culture Microplate, with Lid	Corning	353072	container for growing sorted monoclonal cells
FCS Express	De Novo Software:	N/A	software for characterizing flow cytometry data
Fetal Bovine Serum, Regular, USDA 500 mL	Corning	35-010-CV	reagent for growing mammalian cells
Fisherbrand Petri Dishes with Clear Lid - Raised ridge; 100 x 15 mm	Fisher Scientific	FB0875712	equipment for growing bacteria
Gibco DMEM, High Glucose	Thermo Fisher Scientific	11-965-092	reagent for growing mammalian cells
Hs00932330_m1 KRAS isoform a Taqman Gene Expression Assay	Life Technologies	4331182	qPCR Probe
Hygromycin B (50 mg/mL), 20 mL	Life Technologies	10687-010	reagent for selecting cells with proper gene circuit integration
iScript Reverse Transcription Supermix	Bio-Rad Laboratories	1708890	reagent for converting RNA to cDNA
Laboratory Freezer -20 °C	VWR	76210-392	equipment for storing experimental reagents
Laboratory Freezer -80 °C	Panasonic	MDF-U74VC	equipment for storing experimental reagents
Laboratory Refrigerator +4 °C	VWR	76359-220	equipment for storing experimental reagents
LB Broth (Lennox) , 1 kg	Sigma-Aldrich	L3022-250G	reagent for growing bacteria
LIPOFECTAMINE 3000	Life Technologies	L3000008	reagemt for transfecting gene circuits into mammalian cells
MATLAB 2019	MathWorks	N/A	software for analyzing experimental data
Methanol	Acros Organics	413775000	reagent for immunofluorescence reaction
Microcentrifuge Tubes, Polypropylene 1.7 mL	VWR	20170-333	plasticware container
Mr04097229_mr EGFP/YFP Taqman Gene Expression Assay	Life Technologies	4331182	qPCR Probe
MultiTherm Shaker	Benchmark Scientific	H5000-HC	equipment for bacterial transformation
NanoDrop Lite Spectrophotometer	Thermo Fisher Scientific	ND-NDL-US-CAN	equipment for DNA/RNA concentration measurement
NEB Q5 High-Fidelity DNA polymerase 2x Master Mix	NEB	M0492S	reagent for PCR of gene circuit fragments
NEB10-beta Competent E. coli (High Efficiency)	New England Biolabs (NEB)	C3019H	bacterial cells for amplifying gene circuit of interest
NEBuilder HiFi DNA Assembly Master Mix	New England Biolabs (NEB)	E2621L	reagent for combining gene circuit fragements
Nikon Eclipse Ti-E inverted microscope with a DS-Qi2 camera	Nikon Instruments Inc.	N/A	instrument for quantifying gene expression
NIS-Elements	Nikon Instruments Inc.	N/A	software for characterizing fluorescence microscopy data
oligonucleotides	IDT	N/A	reagent used for PCR of gene circuit components
Panasonic MCO-170 AICUVHL-PA cellIQ Series CO2 Incubator with UV and H2O2 Control	Panasonic	MCO-170AICUVHL-PA	instrument for growing mammalian cells
Paraformaldehyde, 16% Electron Microscopy Grade	Electron Microscopy Sciences	15710-S	reagent
PBS, Dulbecco's Phosphate-Buffered Saline (D-PBS) (1x)	Invitrogen	14190144	reagent for mammalian cell culture,reagent for IF incubation buffer
Penicillin-Streptomycin (10,000 U/mL), 100x	Fisher Scientific	15140-122	reagent for growing mammalian cells
primary ERK antibody	Cell Signaling Technology	4370S	primary ERK antibody for immunifluorescence
primary KRAS antibody	Sigma-Aldrich	WH0003845M1	primary KRAS antibody for immunifluorescence
QIAprep Spin Miniprep Kit (250)	Qiagen	27106	reagent kit for purifying gene circuit plasmids
QIAquick Gel Extraction Kit (50)	Qiagen	28704	reagent kit for purifying gene circuit fragments
QuantStudio 3 Real-Time PCR System	Eppendorf	A28137	equipment for qRT-PCR
Relative Quantification App	Thermo Fisher Scientific	N/A	software for quantifying RNA/cDNA amplificaiton
RNeasy Plus Mini Kit	Qiagen	74134	kit for extracting RNA of engineered mammalian cells
Secondary ERK antibody	Cell Signaling Technology	8889S	secondary ERK antibody for immunifluorescence
secondary KRAS antibody	Invitrogen	A11005	secondary KRAS antibody for immunifluorescence
Serological Pipets 5.0 mL	Olympus Plastics	12-102	reagents used for setting up a variety of chemical reactions
SmartView Pro Imager System	Major Science	UVCI-1200	tool for imaging correct PCR bands
SnapGene Viewer (free) or SnapGene	SnapGene	N/A	software DNA sequence design and analysis
Stage top incubator	Tokai Hit	INU-TIZ	tool for carrying PCR, transformation, or gel extraction reactions
TaqMan Fast Advanced Master Mix	Thermo Fisher Scientific	4444557	reagent for PCR of gene circuit fragments
TaqMan Human GAPD (GAPDH) Endogenous Control (VIC/MGB probe), primer limited, 2500 rxn	Life Technologies	4326317E	qPCR Probe
Thermocycler	Bio-Rad	1851148	tool for carrying PCR, transformation, or gel extraction reactions
VisiPlate-24 Black, Black 24-well Microplate with Clear Bottom, Sterile and Tissue Culture Treated	PerkinElmer	1450-605	plate used for light-induction experiments
VWR Disposable Pasteur Pipets, Glass, Borosilicate Glass Pipet, Short Tip, Capacity=2 mL, Overall Length=14.6 cm	VWR	14673-010	reagent for mammalian cell culture
VWR Mini Horizontal Electrophoresis Systems, Mini10 Gel System	VWR	89032-290	equipment for DNA gel electrophoresis
Flp-In 293	Thermo Fisher Scientific	R75007	Engineered cell line with FRT site