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Optogenetics is a powerful tool that uses light to control cellular behavior. Here we enhance high-throughput characterization of optogenetic experiments through the integration of the LED Illumination Tool for Optogenetic Stimulation (LITOS) with the previously published automated platform Lustro. Lustro enables efficient high-throughput screening and characterization of optogenetic systems. The initial iteration of Lustro used the optoPlate illumination device for light induction, with the robot periodically moving the plate over to a shaking device to resuspend cell cultures. Here, we designed a 3D-printed adaptor, rendering LITOS compatible with the BioShake 3000-T ELM used in Lustro. This novel setup allows for concurrent light stimulation and culture agitation, streamlining experiments. Our study demonstrates comparable growth rates between constant and intermittent shaking of Saccharomyces cerevisiae liquid cultures. While the light intensity of the LITOS is not as bright as the optoPlate used in the previous iteration of Lustro, the constant shaking increased the maturation rate of the mScarlet-I fluorescent reporter used. Only a marginal increase in temperature was observed when using the modified LITOS equipped with the 3D-printed adaptor. Our findings show that the integration of LITOS onto a plate shaker allows for constant culture shaking and illumination compatible with laboratory automation platforms, such as Lustro. 

Optogenetics provides precise control of cellular behavior through genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired range of functionality often requires many design-build-test cycles, which is time-consuming and labor-intensive. To address this, we designed Lustro, a platform which integrates light stimulation with laboratory automation to enable high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. A robotic arm is programmed to move a microwell plate between the devices to stimulate optogenetic strains and measure their response. Here we present a protocol for using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol describes how to set up the components of Lustro, integrating an illumination device with an automation workstation, and provides instruction for programming the illumination device, plate reader, and robot.