Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

The standardized optogenetic protocol is designed for labs interested in using light as the stimulus to control cellular properties in engineered mammalian cell systems. This protocol is easy to implement in labs otherwise unfamiliar with optogenetics and provides methods for precise spatial temporal control of engineered biological systems. This method has a broad range of application in specific research areas where spatiotemporal information may be important including cancer biology, systems biology, and tissue differentiation.

To begin with, select the genetic components to combine bind into a single gene circuit or plasmid. For example, mammalian DNA integration sites, light responsive elements, or functional genes. After assembling and examining all the necessary features such as start codons, regulatory or translated sequences, use any genetic engineering and molecular cloning software, annotate and store the DNA sequences for later use and as a reference.

Validate the primers computationally for plasmid construction through the built-in molecular cloning software features, for example, sequence alignment. To generate a stable cell line, transfect the design gene circuits and the desired cells using a liposome mixture of the gene circuit DNA with appropriate recombinase. After cells are 80 to 100%confluent, freeze the cells 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. About two to three weeks after monoclonal sorting, the wells within the 96-well plate should have foci. When 50 to 60%confluency is achieved, split the cells into a 12-well plate.

Add the firmware to the assembled LPA circuit using a microcontroller programmer. Next, combine 3D printed parts in the assembled circuit board using the mounting plate, the circuit board, the LED spacer, the plate adapter, a 24-well plate, a plate lid, mounting bolts, wing nuts and stacking components. To begin an optogenetic experiment, use the Iris software available on the Tabor Lab website to program an SD card for the LPA and explore appropriate light conditions.

Next, enter intensity values with the technical replicates for the desired experimental outline. Add about 75, 000 cells per well in a 24-well black plate in a total volume of 500 microliters, then place the cells in a humidified incubator with 5%carbon dioxide to allow them to settle for two to six hours. After transferring the cells in the tissue culture hood at the end of the incubation, remove the plastic lid and add an adhesive foil strip to the top of the plate for minimizing the light transfer between the wells so that the light can only enter through the LED located at the bottom of the well.

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. 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 and incubate the cells. After incubation, inside the tissue culture hood, remove the foil strip from the plate and let the plate sit for one to two minutes to prevent condensation from forming on the plastic lid.

Next, put the original plastic lid back on the plate and image the cells with the appropriate phase contrast or fluorescence microscope. After 24 to 72 hours post-induction, followed by trypsinization, transfer the entire contents of each well of about 500 microliters to the labeled tubes equipped with the cell strainer. Then using appropriate flow cytometry instrument software, create a forward and side scatter gate to capture the single cells of the appropriate size and granularity to exclude debris and cellular clumps.

Once the gate is set, capture approximately 10, 000 cells with the appropriate gate and adjust cell number depending on the amount of cellular data required and repeat capturing experimental cells in each tube. After induction and trypsinization, transfer the entire contents of each well of about 500 microliters to the labeled tubes. Centrifuge the cells for five minutes at 400 times G.After discarding the supernatant, move the samples to a chemical fume hood.

Resuspend the cells in 750 to 1, 000 microliters of 4%PFA diluted in PBS and pipette up and down several times to break the cell clumps. After 15 minutes of incubation, add 750 to 1, 000 microliters of PBS and again pipette up and down several times, then pellet down the cells for five minutes at 400 times G at room temperature. After discarding the supernatant, move the samples to a chemical fume hood and resuspend the cells in 750 to 1, 000 microliters of ice cold methanol as demonstrated and allow the cells to sit for 30 minutes in a minus 20 degree Celsius freezer.

After the incubation, add 750 to 1, 000 microliters of PBS while by pipetting up and down several times. Then after centrifuging the cells for five minutes at 400 times G, discard the supernatant and move the samples to a chemical fume hood. Resuspend the cells in 100 microliters or other suitable dilution of labeled primary antibody and pipette up and down six to eight times, then incubate for one hour at room temperature.

Next, add 750 to 1, 000 microliters of the incubation buffer and pipette solution up and down several times, then centrifuge the cells for five minutes at 400 times G.Next, resuspend the cells in 500 microliters of PBS and break the clumps by pipetting up and down several times. Transfer the entire contents of each tube to labeled tubes with cells strainers. Bring the cells to appropriate flow cytometry instruments with lasers of the correct wavelengths.

The optical calibration was performed for the devices. Using the acquired image, compensation values for LED were calculated, background signal was subtracted, and pixel intensity was deduced. The coefficient of variation was minimal between 96 wells.

The calibration software demonstrated the LED variation by location and created a gray scale adjustment so that each LED is normalized to the same intensity. Gene expression was induced in engineered lighter cells via fluorescence microscopy at different light pulse durations on the LPA and the cells were imaged for Brightfield and GFP expression. Using flow cytometry, the cells were induced at different pulse length values and showed a dose response of gene expression quantified at the population level of over fourfold change from uninduced states.

The negative feedback achieved over fivefold gene expression noise reduction as shown by comparing various light intensity values for the lighter system versus vivid or light on. The qPCR analysis showed that the engineered lighter cells expressed RNA levels in a dose-responsive manner with over tenfold induction from uninduced states, matching the flow cytometry quantification. The engineered lighter gene circuit showed increasing amounts of blue light that resulted in increased KRAS protein levels and phosphorylated ERK levels.

Several functional assays may be performed including growth, cell motility, wound healing, proliferation, or invasion assays. These methods could provide specific insights into the mechanisms of cancer biology, tissue differentiation, or drug resistance.