High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro

Different devices such as bioreactors and microwell plate illuminators are currently available. Bioreactors offer fine temporal resolution, but are limited in throughput. Microwell plate devices offer higher throughput, but lack automation for reliable recording of outputs over time.

Lustro offers reliable and high throughput characterization of optogenetic systems in real time. It can also interface with assays that can be performed by liquid handling robots. Optogenetic systems are an attractive tool due to the fine spatial and temporal control they offer over cellular behavior.

The ability to design, build, and test optogenetic systems more rapidly will pave the way for better understanding and control of biological behavior. 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 a 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.