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.

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

Author Spotlight: Advancing Optogenetics Research Using Lustro 

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

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 ...

high-throughput-optogenetics-experiments-yeast-using-automated

Research

JoVE Journal

Bioengineering

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

Video: Author Spotlight: Advancing Optogenetics Research Using Lustro 

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...