This work was supported by National Institutes of Health grant R35GM128873 and National Science Foundation grant 2045493 (awarded to M.N.M.). Megan Nicole McClean, Ph.D. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. Z.P.H. was supported by an NHGRI training grant to the Genomic Sciences Training Program 5T32HG002760. We acknowledge fruitful discussions with McClean lab members, and in particular, we are grateful to Kieran Sweeney for providing comments on the manuscript.

The yeast strains utilized in this study are documented in the Table of Materials. These strains exhibit robust growth within the temperature range of 22 &#176;C to 30 &#176;C and can be cultivated in various standard yeast media.
1. Setting up the automation workstation
<ol>
	<li>Equip the automated workstation with a Robotic Gripper Arm (RGA, see Table of Materials) capable of moving microwell plates (Figure 1).</li>
	<li>Install a microplate heater shaker (see Table of Materials) into the automated workstation (Figure 1(1)) with an automatic plate locking mechanism that provides RGA access.</li>
	<li>Position a microplate reader adjacent to the automated workstation (Figure 1(2)), ensuring RGA accessibility.</li>
	<li>Incorporate a microplate illumination device (Figure 1(3)) that allows convenient RGA access. Examples include the optoPlate8 (as used in this study) or LITOS9 (see Table of Materials).</li>
</ol>
2. Preparation of the illumination device
<ol>
	<li>Construct and calibrate the optoPlate (or alternative illumination device) following established methods8,10,11.</li>
	<li>Utilize an adaptor on the illumination device to enable RGA access.</li>
	<li>Program the optoPlate using a spreadsheet input10 or a graphical interface12. Follow step 3 to execute the light stimulation programs.</li>
</ol>
3. Designing a light stimulation program
<ol>
	<li>Determine the desired light conditions for the experiment.</li>
	<li>Enter the desired light conditions, including light intensity, light start time, pulse length, pulse number, and interpulse duration, into a spreadsheet. 
	NOTE: Flash this information onto the optoPlate using the instructions provided in the GitHub repository: github.com/mccleanlab/Optoplate-96. Remember that the sample plate will not be illuminated while in the microplate reader or on the heater shaker. The duration and frequency of these events may require optimization based on specific experimental requirements.</li>
	<li>Include dark conditions for each strain to enable proper background measurements.</li>
	<li>For initial characterization experiments to assess transformant functionality, use a high light intensity. 
	&#8203;NOTE: The light intensity should be optimized for more sensitive experiments, as excessive light can be phototoxic to yeast13.</li>
</ol>
4. Preparation of the microplate reader
<ol>
	<li>Configure the microplate reader to measure the desired quantity of interest before conducting experiments. 
	NOTE: In this example, the microplate reader was configured to measure fluorescence from a reporter expressed by the strain of interest. Other outputs, such as luminescence or optical density, can be used depending on the experimental requirements.</li>
	<li>Cultivate the strain of interest (along with a nonfluorescent control) in synthetic complete (SC) media14 (or another low fluorescence media) (see Table of Materials) to the highest cell density to be measured. Transfer the cultures into a glass-bottom black-walled microwell plate (see Table of Materials) and measure them to determine the optimal microplate reader settings. 
	NOTE: Ensure accurate readings by correctly entering the plate dimensions and measuring the plate from below.</li>
	<li>Refer to a fluorescent protein database (for example, fpbase.org) to obtain approximate absorption and emission spectra for the target fluorescent protein15.</li>
	<li>Determine the z-value (distance between the plate and reader) by performing a z-scan on wells containing the fluorescent and nonfluorescent strains. Select the z-value&#160;that yields the highest signal-to-noise ratio.</li>
	<li>Optimize the absorption and emission spectra by conducting absorption and emission scans on the fluorescent and nonfluorescent strains to determine the optimal signal-to-noise ratio.</li>
	<li>Measure the fluorescent strain with the gain set to the optimal level, determining the highest optical gain that can be used without resulting in an overflow measurement error. 
	&#8203;NOTE: This optical gain should be manually set consistently across experiments involving the same strain to ensure consistency of the results.</li>
	<li>Prepare a measurement script (refer to Figure 2) in the microplate reader software. This script configures the instrument to measure the optical density of the cultures and the fluorescence spectra of any fluorescent proteins to be measured. 
	&#8203;NOTE: Measure the optical density of strains at 600 nm unless they express red fluorescent proteins. For strains expressing red fluorescent proteins, measure the optical density at 700 nm to avoid bias16.</li>
	<li>Set the measurement script to maintain an internal incubation temperature of 30 &#176;C during measurements.</li>
	<li>Configure the measurement script to export the data into a spreadsheet or ASCII files, according to preference.</li>
</ol>
5. Programming the robot
<ol>
	<li>Configure the worktable definition in the automated workstation software based on the physical layout of carriers (e.g., heater shakers, nest platforms, illumination devices) and the labware (i.e., the 96-well plate) (see Table of Materials). Create a script in the automation workstation software to execute light induction and measurements (Figure 3), following the below steps.</li>
	<li>Disable any internal illumination sources to prevent background activation of optogenetic systems.</li>
	<li>Set the heater shaker to maintain a temperature of 30 &#176;C. When the plate is not on the heater shaker, it will be at ambient temperature (22 &#176;C).</li>
	<li>Utilize loops, a timer, and a loop counting variable to repeat the steps of inducing and measuring the cells at regular intervals.</li>
	<li>Before recording measurements, shake the sample plate to ensure proper suspension of all cells. A 60 s shake at 1,000 rpm with a 2 mm orbital is generally sufficient to resuspend S288C S. cerevisiae cells and avoid measurement bias.</li>
	<li>Move the sample plate to the microplate reader using the robot arm and remove the lid (if applicable) to prevent bias in optical density measurement. The control software will automatically remove and replace the lid to the designated position&#160;if the microplate reader carrier definition is set to not allow lids.</li>
	<li>Execute the microplate reader measurement script as described in step 4.</li>
	<li>Replace the lid on the sample plate and move the plate to the illumination device using the robot arm.</li>
	<li>Set the script to wait until the timer reaches the specified time interval (e.g., 30 min multiplied by the loop counting variable) and then repeat the entire loop for the desired number of iterations (e.g., 48 times for a 24 h experiment).</li>
	<li>Troubleshoot potential errors and ensure the proper functioning of the carrier and labware definitions, as well as the RGA&#39;s ability to accurately pick up and place the plate by running an empty plate through the script loop multiple times.</li>
	<li>Configure user alerts to notify the user of any instrument state changes or errors that may occur during the execution of the protocol.</li>
</ol>
6. Setting up the sample plate
<ol>
	<li>Grow yeast strains on rich media plates, such as YPD agar17 (see Table of Materials). Include a nonfluorescent (negative) control.</li>
	<li>Select colonies from the plates and inoculate them into 3 mL of SC media14 (or another low fluorescence media, such as LFM18) in glass culture tubes. Incubate the cultures overnight at 30 &#176;C on a roller drum while keeping them in the dark or under non-responsive light conditions (e.g., red light for blue light-responsive systems).</li>
	<li>Measure the optical density of the overnight cultures by diluting 200 &#181;L of each culture into 1 mL of SC media and recording the optical density at 600 nm (OD600) using a spectrophotometer or microplate reader.</li>
	<li>Dilute each overnight culture to an OD600 of 0.1 in glass culture tubes. For higher throughput strain testing, perform automated dilutions in microwell plates.</li>
	<li>Pipette the diluted cultures into the 96-well plate. Include triplicate wells for each condition (i.e., three identical wells with the same strain and light condition) to account for technical variation. Include wells with blank media and nonfluorescent cells as negative controls to measure background fluorescence and optical density.</li>
	<li>Incubate the plate at 30 &#176;C with shaking for 5 h before commencing the light induction experiment. Adjust the dilution amounts and incubation times as necessary to optimize for specific strains and experimental conditions.</li>
</ol>
7. Performing the experiment
<ol>
	<li>Place the sample plate onto the heater shaker and initiate the automation script as outlined in step 5.</li>
	<li>Commence the light stimulation program as described in step 3 once the initial measurement on the plate reader has been recorded.</li>
	<li>If the automation workstation is not in a dark room, cover it with a blackout curtain to avoid background illumination.</li>
</ol>
8. Data analysis
<ol>
	<li>Create a spreadsheet map for the experiment, reflecting the 8 x 12 layout of the 96-well plate. Ensure that the map includes the names of the strains being measured in one grid and descriptions of the light conditions used in another grid.</li>
	<li>Utilize a Python script or another preferred programming language to analyze the exported data. Import the map spreadsheet into an array, associating the strain names and condition names with each well of the plate. 
	NOTE: Sample code for analysis can be found at: https://github.com/mccleanlab/Lustro. Alternatively, one can employ an application to parse data from various plate readers19.</li>
	<li>Read the data from the exported experiment spreadsheet into another array.</li>
	<li>Generate plots of the optical density values and fluorescence values for each strain and condition over time, as illustrated in Figure 4.</li>
	<li>Examine the optical density plots to determine the stages of exponential growth or saturation in the cultures. This information will aid in selecting appropriate timepoints for comparing fluorescence measurements between strains or conditions, as depicted in Figure 5.</li>
</ol>

Yeast Optogenetic Response Analysis Using the Lustro Automation Technique

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+characterization+methods+overcome+key+challenges+in+synthetic+and+systems+biology.">Optogenetic characterization methods overcome key challenges in synthetic and systems biology.</a> Nature Chemical Biology. 10, 502-511 (2014).</li><li>Hallett, R. A., Zimmerman, S. P., Yumerefendi, H., Bear, J. E., Kuhlman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+in+vitro+and+in+vivo+Activities+of+Light+Inducible+Dimers:+a+Cellular+Optogenetics+Guide.">Correlating in vitro and in vivo Activities of Light Inducible Dimers: a Cellular Optogenetics Guide.</a> ACS Synthetic Biology. 5 (1), 53-64 (2016).</li><li>Scott, T. D., Sweeney, K., McClean, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+signal+generators:+integrating+synthetic+biology+tools+and+in+silico+control.">Biological signal generators: integrating synthetic biology tools and in silico control.</a> Current Opinion in Systems Biology. 14, 58-65 (2019).</li><li>Harmer, Z. P., McClean, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lustro:+High-throughput+optogenetic+experiments+enabled+by+automation+and+a+yeast+optogenetic+toolkit.">Lustro: High-throughput optogenetic experiments enabled by automation and a yeast optogenetic toolkit.</a> ACS Synthetic Biology. 12 (7), 1943-1951 (2023).</li><li>Bindels, D. 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The authors have nothing to disclose.

The Lustro protocol presented here automates the culturing, illumination, and measurement processes, enabling high-throughput screening and characterization of optogenetic systems6. This is achieved by integrating an illumination device, microplate reader, and shaking device into an automation workstation. This protocol specifically demonstrates Lustro&#39;s utility for screening different optogenetic constructs integrated into the yeast S. cerevisiae and comparing light induction program...

Optogenetics is a powerful technique that utilizes light-sensitive proteins to control the behavior of cells with high precision1,2,3. However, prototyping optogenetic constructs and identifying optimal illumination conditions can be time-consuming, making it difficult to optimize optogenetic systems4,5. High-throughput methods to rapidly screen and characterize the activity of optogenetic systems can accelerate the design-build-test cycle for prototyping constructs and exploring their function.
The Lustro platform was developed as a laboratory automation technique designed for high-throughput screening and characterization of optogenetic systems. It integrates a microplate reader, illumination device, and shaking device with an automation workstation6. Lustro combines automated culturing and light stimulation of cells in microwell plates (Figure 1 and Supplementary Figure 1), enabling the rapid screening and comparison of different optogenetic systems. The Lustro platform is highly adaptable and can be generalized to work with other laboratory automation robots, illumination devices, plate readers, cell types, and optogenetic systems, including those responsive to different wavelengths of light.
This protocol demonstrates the setup and use of Lustro for characterizing an optogenetic system. Optogenetic control of split transcription factors in yeast is used as an example system to illustrate the function and utility of the platform by probing the relationship between light inputs and the expression of a fluorescent reporter gene, mScarlet-I7. By following this protocol, researchers can streamline the optimization of optogenetic systems and accelerate the discovery of new strategies for the dynamic control of biological systems.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well glass bottom plate with&#160; #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>P96-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>BioShake 3000-T elm (heater shaker)</td>
			<td>QINSTRUMENTS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluent Automation Workstation</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LITOS (alternative illumination device)</td>
			<td>Hohener, et al. Scientific Reports. 2022</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>optoPlate-96 (illumination device)</td>
			<td>Bugaj, et al. Nature Protocols. 2019</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Robotic Gripper Arm</td>
			<td>Tecan</td>
			<td></td>
			<td>Standard or long Z axes; regular gripper head or automatic Finger Exchange System gripper head, both with a choice of gripper fingers &#8211; eccentric, long eccentric, centric, tube; barcode reader option</td>
		</tr>
		<tr>
			<td>Spark (plate reader)</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic Complete media</td>
			<td>SigmaAldrich</td>
			<td>Y1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Connect (user alert app)</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yMM1734 (BY4741 Mat&#945; ura3&#916;0::5&#39; Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagB-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#39; homology&#160; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td>
			<td>Harmer, et al. ACS Syn Bio. 2023</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yMM1763 (BY4741 Mat&#945; ura3&#916;0::5&#39; Ura3 homology, pRPL18B-Gal4DBD-CRY2(535)-tENO1, pRPL18B-Gal4AD-CIB1-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#39; homology&#160; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td>
			<td>Harmer, et al. ACS Syn Bio. 2023</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yMM1765 (BY4741 Mat&#945; ura3&#916;0::5&#39; Ura3 homology, pRPL18B-Gal4DBD-eMagA-tENO1, pRPL18B-eMagBM-Gal4AD-tENO1, pGAL1-mScarlet-I-tENO1, Ura3, Ura 3&#39; homology&#160; his3D1 leu2D0 lys2D0 gal80::KANMX gal4::spHIS5)</td>
			<td>Harmer, et al. ACS Syn Bio. 2023</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>YPD Agar</td>
			<td>SigmaAldrich</td>
			<td>Y1500</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput optogenetics experiments in yeast using the automated platform lustro

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient 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. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of&#160;their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro&#39;s components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Author Spotlight: Advancing Optogenetics Research Using Lustro 

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 liqui-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.

Figure 4A shows the fluorescence values over time for an optogenetic strain expressing a fluorescent reporter controlled by a light-inducible split transcription factor. The different light conditions used in the experiment are reflected by variations in the duty cycle, which represents the percentage of time the light is on. The overall fluorescence level is observed to be proportional to the duty cycle of the light stimulation. Figure 4B displays the correspon...

Watch this Scientific Journal Video about Advancing Optogenetics Research Using Lustro at JoVE.com

This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in yeast.

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to ...

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Research

JoVE Journal

Bioengineering

501 Views.  University of Wisconsin-Madison. 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 liqui-handling robots. Optogenetic systems are an a...