We thank Jozsef Stork, Mitchel Richmond, Jarrad Gollihue, and Andrea Sanchez for constructive and critical discussion. Dr. Sharyn Perry for the phenotypic photographs. This material is based upon work supported by the National Science Foundation under Cooperative Agreement No. 1355438.

1. Creating a Dilution Library
<ol>
	<li>Label 625 Dilution Library plates by hand, ensuring that they match to their corresponding plate from the chemical library. Additionally, connect in flow and out flow hoses to the Multichannel Tip Wash Automated Labware Positioner (ALP) by passing them through the Console Drive to the 5 Gallon Reservoir (see Table of Materials).</li>
	<li>Access the computer and turn on the wash pump through the connection of the Device Controller to the Multichannel Tip Wash ALP in order to circulate water. This will turn off automatically at the end of the protocol.</li>
	<li>Load, by hand, the Stacker 10&#39;s attached to the Stacker Carousel, in the following order in Hotels A - D (Figure 4, Stacker); one box of AP96 P20 Pipette Tips in Room 1, four 96-Well V-Bottom Plates in Rooms 2 - 5 with the two upper plates containing stock concentrations from the ordered library and the two lower&#160;plates empty (Figure 5, Stacker). Additionally, load one box of AP96 P20 Pipette Tips in Room 6, and four 96-Well V-Bottom Plates in Rooms 7 - 9 with the two upper&#160;plates containing stock concentrations of the ordered library and the two lower plates empty (Figure 5, Stacker).</li>
	<li>Set up, by hand, the deck with a 300 mL water reservoir on P3, a 300 mL 70% EtOH bath on P7, Tip Loader ALP (TL1), and Multichannel Tip Wash ALP (TW1) (Figure 4, Deck and Figure 5, Deck).</li>
	<li>Using the operating software, present AP96 P20 Pipette Tips from the Stacker 10 and move them to the Tip Loader ALP. 
	NOTE: 1.5 through 1.12 all are done with the liquid handling robot&#39;s operating software; see Table of Materials.</li>
	<li>Present Room 2 from Hotel A and separate all four 96-Well V-Bottom Plates on the deck, placing the bottom two on P4 and P8 and the top two on P5 and P9 (Figure 4).</li>
	<li>Load AP96 P20 Pipette Tips with the Tip Loader ALP onto the 96-Channel 200 &#181;L Head. Aspirate 90 &#181;L from the 300 mL water reservoir and dispense into the 96-Well V-Bottom Dilution Plate on P4. Repeat this step for the plate on P8.</li>
	<li>Mix the chemical library plate on P5&#160;by repetitively aspirating and dispensing 15 &#181;L three times. Additionally, aspirate 10 &#181;L from the chemical library plate on P5&#160;and dispense 10 &#181;L into the dilution plate on P4.</li>
	<li>Mix the solutions of the plate on P4&#160;by repetitively aspirating and dispensing 50 &#181;L a total of three times. Once mixed, clean the AP96 P20 Pipette Tips by aspirating and dispensing 70 &#181;L of 70% EtOH from P7, then washing them in the Multichannel Tip Wash ALP by aspirating and dispensing a 110% volume of water four times.</li>
	<li>Repeat steps 1.8 - 1.9 for the second pair of plates on P8 and P9. Upon creating the second 96-Well V-Bottom Dilution Plate, stack the plates in the following order from bottom to top: P9, P5, P8, and P4. Then, place the stack on one empty Static ALP; either P1, P2, P6, P10, P11, P12, or P13.</li>
	<li>Repeat steps 1.6 - 1.10 until Room 5 in Hotel A is empty. Repeat step 1.5 upon reaching Room 6, moving new AP96 P20 Pipette Tips to the Tip Loader ALP and placing the used AP96 P20 Pipette Tips on an empty Static ALP.</li>
	<li>Repeat steps 1.6 - 1.10 until Room 9 of Hotel A is empty. However, in order to proceed to Hotel B, the plates and tips on the deck must be reloaded into Hotel A.</li>
	<li>Re-fill, by hand, the 300 mL water reservoir. This step is crucial, and the computer program can incorporate a pause detailing this message, requiring the user to hit &#39;continue&#39;, before carrying out the next step.</li>
	<li>Repeat steps 1.5 - 1.13 for the remaining hotels, ensuring a full 300 mL water reservoir each time prior to proceeding to the next hotel.</li>
</ol>
2. Adding Media-seed Mixture to Screening Plates
<ol>
	<li>Make &#189; Murashige and Skoog (MS) Media with 0.1% Agar by the addition of 4.3 g MS Salts, 0.50 g MES, 1.0 g Agar to 1 L DI H2O. Adjust the pH to 5.7 though the addition of 5 M potassium hydroxide while monitoring with a pH probe.</li>
	<li>Sterilize seeds by shaking them in 1% bleach and SDS between 15 and 30 min, and then rinse 4 times with an equal volume of water by centrifugation. Once seeds are sterile, place them at 4 &#176;C from 24 hours to 7 days for vernilization. The Arabidopsis Biological Resource Center describes additional methods of sterilization, vernilization, and growth22.</li>
	<li>Add seeds to media by hand at a density of 0.1 g/100 mL. This density results in an average of 3 - 10 seeds per well of a 96-well plate.</li>
	<li>Place, by hand, four 96-Well Flat-Bottom Plates in Rooms 1 and 2 of Hotel A (Figure 6, Hotel A). Place a box of AP96 P250 Pipette Tips on the Tip Loader ALP, a 300 mL reservoir filled with the media-seed mixture created in steps 2.1 - 2.3 on P3, and a 300 mL reservoir filled with 70% EtOH on P7 (Figure 4, Deck and Figure 6, Deck). 
	NOTE: 2.5 through 2.8 are done with the operating software.</li>
	<li>Present Rooms 1 and 2 in Hotel A, and separate the stacks of four plates. Place one plate on each of the empty static ALPs (P4, P5, P6, P8, P9, P10, P11, and P12). Load AP96 P250 Pipette Tips on the 96-Channel 200 &#181;L Head.</li>
	<li>Aspirate 90 &#181;L from the 300 mL media-seed reservoir on P3 and dispense into the first 96-Well Flat-Bottom Plate. Repeat this process until all eight plates contain the media-seed mixture.</li>
	<li>Clean the AP96 P250 Pipette Tips by aspirating and dispensing 70 &#181;L from the 300 mL reservoir filled with 70% EtOH on P7. Wash the tips in the Multichannel Tip Wash ALP by aspirating and dispensing a 110% volume of water four times, unload the tips at TL1, and gather the plates by hand.</li>
</ol>
3. Adding Small Molecules to Screening Plates
<ol>
	<li>Load, by hand, a box of AP96 P250 Pipette Tips into Room 1 of Hotel A, two 96-Well V-Bottom Dilution Library Plates into Rooms 2, 4, 6, and 8, and two 96 Well Flat-Bottom Screening Plates into Rooms 3, 5, 7, and 9 (Figure 4, Stacker and Figure 7, Hotel A). Additionally, connect hoses to and from the Multichannel Tip Wash ALP to the 5 Gallon Reservoir. 
	NOTE: 3.2 through 3.10 are done with the operating software.</li>
	<li>Configure the deck to contain a 300 mL 70% EtOH wash reservoir at P7; the media-seed reservoir can be left on the deck at P3 (Figure 4, Deck and Figure 7, Deck). Additionally, turn on the Console Drive through connection of the Device Controller to circulate water through the Multichannel Tip Wash ALP. This will turn off automatically at the end of the protocol.</li>
	<li>Present the AP96 P250 Pipette Tip Box from Hotel A and move it to the Tip Loader ALP.</li>
	<li>Present the 96-Well V-Bottom Dilution Library Plates from Room 2 of Hotel A to the deck and place one on Static ALP P4 and one on P8. Present the 96-Well Flat-Bottom Screening Plates from room 3 of Hotel A to the deck and place one on Static ALP P5 and one on P9.</li>
	<li>Load AP96 P250 Pipette Tips with the Tip Loader ALP onto the 96-Channel 200 &#181;L Head.</li>
	<li>Mix the 96-Well V-Bottom Dilution Plate on P4 by aspirating and dispensing 50 &#181;L three times. Following that, aspirate 10 &#181;L from this plate and dispense into the 96-Well Flat-Bottom Screening Plate on P5.</li>
	<li>Mix the solutions in the plate at P5 by aspirating and dispensing 50 &#181;L three times. Clean the AP96 P250 Pipette Tips with ethanol by aspirating and dispensing 70 &#181;L of 70% EtOH from the reservoir at P7 and then wash the tips in the Multichannel Tip Wash ALP by aspirating and dispensing a 110% volume of water four times.</li>
	<li>Repeat steps 3.5 and 3.6 for the second 96-Well V-Bottom Dilution Library Plate (P8) and 96-Well Flat-Bottom Screening Plate (P9).</li>
	<li>Stack the two 96-Well V-Bottom Dilution Library Plates together and the two 96-Well Flat Bottom Screening Plates together. Move the plates to Static ALPs P1, P2, P6, P10, P11, P12, or P13.</li>
	<li>Repeat steps 3.4 - 3.9 three times, adding diluted chemicals to screening plates a total of eight times. Finally, check the number of seeds in each well of the screening plates through visual conformation and supplement those wells with fewer than three seeds by additional sterilized and vernalized seed.</li>
</ol>
4. Incubation and Visualization of Screening Plates
<ol>
	<li>Incubate the 96-well Flat-Bottom Screening Plates for four days in an environmental chamber at 22 &#176;C on a 16/8 light/dark cycle in a desiccation proof container. Visualize the 96-well Flat-Bottom Screening Plates under a dissecting microscope. Record all aberrant phenotypes for further investigation.</li>
</ol>

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The authors declare that they have no competing financial interests.

This protocol is designed to aid researchers in accomplishing a forward chemical genetics screen on Arabidopsis. We provide representative results from a screen of 50,000 compounds (Figure 2 and Figure 3), one of the largest forward chemical genetics screens performed on Arabidopsis to date9,13,23. The use of a liquid handling robot enabled more efficient dilution libra...

In the past 20 years researchers in the field of plant biology have made great strides using chemical genetics approaches, both forward and reverse, improving our understanding of cell wall biosynthesis, the cytoskeleton, hormone biosynthesis and signaling, gravitropism, pathogenesis, purine biosynthesis, and endomembrane trafficking1,2,3,4,5. Employing forward chemical genetics techniques enables the identification of phenotypes of interest and allows researchers to understand the genotypic underpinnings of particular processes. Conversely, reverse chemical genetics seeks out chemicals that interact with a pre-determined protein target6. Arabidopsis has been at the forefront of these discoveries in plant biology because its genome is small, mapped, and annotated. It has a short generation time, and there are multiple mutant/reporter lines available to facilitate the identification of aberrant subcellular machinery7.
There are two major bottlenecks that slow the progress of forward chemical genetic screens, the initial screening process and determining the target of the compound of interest8. A major aid in increasing the speed of small molecule selection is the use of automation and automated equipment9. Liquid handling robots are an excellent tool for handling large libraries of small molecules and have been instrumental in driving progress in the biological sciences10. The protocol presented here is designed to alleviate the bottleneck associated with the screening process, enabling the identification of bioactive small molecules at a rapid rate. This technique decreases the burden of labor and time on behalf of the operator while also decreasing the economic cost to the principle investigator.
Thus far, most chemical libraries analyzed have held between 10,000 and 20,000 compounds, some with as many as 150,000 and some with as few as 709,11,12,13,14,15,16. The protocol introduced herein was implemented on a small molecule library of 50,000 compounds (see Table of Materials), one of the larger forward chemical genetics screens conducted on Arabidopsis to date. This protocol fits with the current trend towards increased efficiency and speed regarding forward chemical genetics, especially as it pertains to herbicide discovery, insecticide discovery, fungicide discover, drug discovery, and cancer biology17,18,19,20,21. Though implemented here with Arabidopsis, this protocol, could easily be adapted to cell cultures, spores, and potentially even insects in liquid medium within 96-, 384-, or 1536-well plates. Due to its small size, Arabidopsis is amenable to screening in 96 well plates. However, distributing seeds evenly among wells is a challenge. Hand seeding is accurate but labor intensive, and though there are devices designed to dispense seeds into 96-well plates, they are expensive to purchase. Here, we show how this step can be circumvented with just a small loss in accuracy.
The overall goal of this method was to make screening a large chemical library against Arabidopsis more manageable, without compromising accuracy, via the use of a liquid handling robot. The use of this method improves the efficiency of the researcher by decreasing the time taken to complete initial dilution series management and subsequent phenotypic screens, allowing quick visualization of samples under a dissecting microscope, and rapid identification of novel bioactive small molecules. Figure 1 depicts this protocol&#39;s key outcomes in 4 steps.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57393/57393fig1.jpg" /> 
Figure 1: Overall workflow of the forward chemical genetics screen. An overview of the protocol to be described with some detail for each of the 4 key steps. 1: Receiving the Chemical Library, 2: Making the Dilution Library, 3: Making the Screening Plates, and 4: Incubating and visualizing the Screening Plates. <a href="/files/ftp_upload/57393/57393fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Keyboard</td><td>Local Provider</td><td>N/A</td><td>Used for protocol design and operating the Biomek FX</td></tr><tr><td>Mouse</td><td>Local Provider</td><td>N/A</td><td>Used for protocol design and operating the Biomek FX</td></tr><tr><td>Computer Screen</td><td>Local Provider</td><td>N/A</td><td>Used for protocol design and operating the Biomek FX</td></tr><tr><td>Computer</td><td>Local Provider</td><td>N/A</td><td>Used for protocol design and operating the Biomek FX</td></tr><tr><td>DIVERSet Diverse Screening Library</td><td>ChemBridge</td><td>N/A</td><td>Chemical library</td></tr><tr><td>Biomek Software</td><td>Beckman Coulter</td><td>N/A</td><td>Runs and designs the Biomek FX</td></tr><tr><td>Device Controller</td><td>Beckman Coulter</td><td>719366</td><td>Operates the water pump/tip washing station</td></tr><tr><td>Stacker Carousel Pendent</td><td>Beckman Coulter</td><td>148240</td><td>Manual operation of Biomek Stacker Carousel</td></tr><tr><td>Biomek Stacker Carousel</td><td>Beckman Coulter</td><td>148520</td><td>Rotary unit that houses all FX Stacker 10&#039;s</td></tr><tr><td>FX Stacker 10</td><td>Beckman Coulter</td><td>148522</td><td>Elevator unit that houses components for screen</td></tr><tr><td>FX Stacker 10</td><td>Beckman Coulter</td><td>148522</td><td>Elevator unit that houses components for screen</td></tr><tr><td>FX Stacker 10</td><td>Beckman Coulter</td><td>148522</td><td>Elevator unit that houses components for screen</td></tr><tr><td>FX Stacker 10</td><td>Beckman Coulter</td><td>148522</td><td>Elevator unit that houses components for screen</td></tr><tr><td>Biomek FX</td><td>Beckman Coulter</td><td>https://www.beckman.com/liquid-handlers</td><td>Robot that performs the desired operations</td></tr><tr><td>Accuframe</td><td>Artisan Technology Group</td><td>76853-4</td><td>Frames arm to place components corretly</td></tr><tr><td>Framing Fixture</td><td>Beckman Coulter</td><td>719415</td><td>Centers arm in the Accuframe</td></tr><tr><td>Multichannel Tip Wash ALP</td><td>Beckman Coulter</td><td>719662</td><td>Washes the tips after the ethanol bath</td></tr><tr><td>Tip Loader ALP</td><td>Beckman Coulter</td><td>719356</td><td>Pneumatically loads tips onto the arm</td></tr><tr><td>Air Compressor</td><td>Local Provider</td><td>N/A</td><td>Provides air for pneumatic tip loading</td></tr><tr><td>MasterFlex Console Drive</td><td>Cole-Parmer</td><td>77200-65</td><td>Pump used to circulate water through the Multichannel Tip Washer</td></tr><tr><td>Air Hose</td><td>Local Provider</td><td>N/A</td><td>Provides air from air compressor to Tip Loader</td></tr><tr><td>Water Hose</td><td>Local Provider</td><td>N/A</td><td>Provides water from 5 Gallon Reserviour to Tip Washer</td></tr><tr><td>Static ALP&#039;s</td><td>Beckman Coulter</td><td>Comes with Biomek FX</td><td>Supports equipment for the Screen</td></tr><tr><td>5 Gallon Reserviour</td><td>Local Provider</td><td>N/A</td><td>Recirculates the dirty water from cleaning the tips</td></tr><tr><td>Grippers</td><td>Beckman Coulter</td><td>Comes with Biomek FX</td><td>Grabs and moves the equipment to the correct places</td></tr><tr><td>96-Channel 200 &amp;micro;L Head</td><td>Beckman Coulter</td><td>Comes with Biomek FX</td><td>Holds the 96 tips used within the screen</td></tr><tr><td>AP96 P200 Pipette Tips</td><td>Beckman Coulter</td><td>717251</td><td>Used to make the screening library</td></tr><tr><td>96 Well Flat Bottom Plate</td><td>Costar</td><td>9018</td><td>Aids in visulization of screen</td></tr><tr><td>96 Well V-Bottom Plate</td><td>Costar</td><td>3897</td><td>Aids in storing of dilution library</td></tr><tr><td>AlumaSeal 96 Sealing Film</td><td>MedSci</td><td>F-96-100</td><td>Seals for storage both the chemicle library and dilution library</td></tr><tr><td>Plastic ziplock sandwich bags</td><td>Local Provider</td><td>N/A</td><td>Used to ensure a humid environment for screen</td></tr><tr><td>AP96 P20 Pipette Tips</td><td>Beckman Coulter</td><td>717254</td><td>Used in the dilution library creation</td></tr><tr><td>Growth Chamber</td><td>Percival</td><td>AR36L3</td><td>Germinates seeds for phenotypic visulization</td></tr><tr><td>Spatula</td><td>Local Provider</td><td>N/A</td><td>Holds seeds to add into wells where liquid seeding failed seed adequatly</td></tr><tr><td>Toothpick</td><td>Local Provider</td><td>N/A</td><td>Pushes seeds from spatula to wells</td></tr><tr><td>Murashige and Skoog Basal Salt Mixture</td><td>PhytoTechnology Laboratories</td><td>M524</td><td>Add to MS media mixture</td></tr><tr><td>MES Free Acid Monohydrate</td><td>Fisher Scientific</td><td>ICN19483580</td><td>Added to MS media to decrease pH</td></tr><tr><td>Agar Powder</td><td>Alfa Aesar</td><td>9002-18-0</td><td>Increases thickness of media to support seed suspension</td></tr><tr><td>5M KOH</td><td>Sigma-Aldrich</td><td>484016</td><td>Increases pH to adequate levels</td></tr><tr><td>1L Media Storage Bottle</td><td>Corning</td><td>1395-1L</td><td>Holds enough media for a screen</td></tr><tr><td>Polypropylene Centrifuge Tubes</td><td>Corning</td><td>431470</td><td>Sterilizes seeds prior to vernilization</td></tr><tr><td>pH Probe</td><td>Davis Instruments</td><td>YX-58825-26</td><td>Used for making media</td></tr><tr><td>ALPs (Automated Labware Positioners) Users Manual</td><td>Beckman Coulter</td><td>PN 987836</td><td>Aids in setting up the accompaning equipment for the Biomek FX</td></tr><tr><td>Biomek 2000 Stacker Carousel Users Guide</td><td>Beckman Coulter</td><td>609862-AA</td><td>Aids in setting up the Stacker Carousel</td></tr><tr><td>Biomek FX and FX&lt;sup&gt;P&lt;/sup&gt; Laboratory Automation Workstations Users Manual</td><td>Beckman Coulter</td><td>PN 987834</td><td>Used to frame the Multichannel Pod</td></tr><tr><td>Biomek FXP Laboratory Automation Workstation Customer Startup Guide</td><td>Beckman Coulter</td><td>PN B32335AB</td><td>Used to aid in setting up the Biomek FX</td></tr><tr><td>Biomek Software User&#039;s Manual</td><td>Beckman Coulter</td><td>PN 987835</td><td>Used to set up and understand the Software</td></tr></tbody></table>

optimizing the use of a liquid handling robot to conduct a high throughput forward chemical genetics screen of arabidopsis thaliana

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

The ability to accurately and efficiently characterize phenotypes based on the addition of small molecules at screening concentrations under a dissecting microscope is the end goal of this method of forward chemical genetics on Arabidopsis. The phenotypes observed when all 50,000 compounds had been screened was diverse and could be broken into several distinct classes (Figure 2). Figure 3A-F depicts exam...

Watch this Scientific Journal Video about Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana at JoVE.com

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology.

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

optimizing-use-liquid-handling-robot-to-conduct-high-throughput

Research

JoVE Journal

Bioengineering

6.6K Views.  University of Kentucky. A high throughput screen of synthetic small molecules was conducted on the model plant species, Arabidopsis thaliana. This protocol, developed for a liquid handling robot, increases the speed of forward chemical genetics screens, accelerating the discovery of novel small molecules affecting plant physiology.

Video: Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Immunology and Infection

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Biology

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Summary

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High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

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