Start by setting up the automation workstation. Incorporate a microplate illumination device, such as an optoPlate, that allows convenient robot gripper arm access. Next, process a spreadsheet with the available MATLAB script to program the optoPlate.
Execute the light stimulation program by flashing onto the optoPlate. To program the robot, troubleshoot any potential errors to ensure the proper functioning of the carrier and LabWare definitions. Run an empty plate through the script loop multiple times to verify the robot gripper arm's ability to pick up and place the plate accurately.
Next, select colonies from the plates to set up the sample plate. Then inoculate them into three milliliters of synthetic complete media in glass culture tubes. Incubate these cultures overnight at 30 degrees Celsius on a roller drum in the dark.
In cuvette, dilute 200 microliters of the culture in one milliliter of synthetic complete medium. Record its optical density using a spectrophotometer. Next, dilute each overnight culture to an optical density of 0.1 in glass culture tubes.
Pipette the diluted cultures into a 96-well plate. Include wells with blank media and non-fluorescent cells as negative controls. Incubate the sample plate in a shaker incubator at 30 degrees Celsius for five hours.
Now, place the sample plate on the heater shaker. Initiate the automation script by clicking on Run. Once the initial measurement has been recorded on the plate reader, start the light stimulation program by switching on the power strip of the illumination device.
The fluorescence values over time for an optogenetic strain showed that the overall fluorescence level was proportional to the duty cycle of the light stimulation. The corresponding optical density values were consistent, suggesting that growth remained unaffected by varying light conditions. Different optogenetic strains employed different responses to light pulse intensity, period, and duty cycle variations.
