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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.
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Optogenetic Tools for Control of Public Goods in Saccharomyces cerevisiae
Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions.
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- Award ID(s):
- 2045493
- PAR ID:
- 10313639
- Date Published:
- Journal Name:
- mSphere
- Volume:
- 6
- Issue:
- 4
- ISSN:
- 2379-5042
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/mSphere.00581-21
