Name: optimizing the use of a liquid handling robot to conduct a high throughput forward chemical genetics screen of arabidopsis thaliana
Uploaded: 2018-04-30T13:30:00
Description: 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

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 AL...

<ol>
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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&#39;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&#39;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 &#181;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 FXP 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&#39;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 ...

The overall goal of this protocol is to perform a high throughput forward chemical genetics screen on Arabidopsis seedlings. With this method, we can efficiently and effectively discover novel small molecules that affect plant physiology. These discoveries will provide the foundation for further studies into physiological processes, such as cell wall synthesis.The main advantage of this technique is that tens of thousands of small molecules can be screened in a short period of time. Though this protocol is designed for the model plant species Arabidopsis, it can easily be adapted to other organisms, such as insects, microorganisms, or cell culture. We first had the idea for this protocol when we learned that the university's genomic center needed to find a home for a surplus liquid-handling robot.We immediately volunteered to take it off their hands. Prepare the dilution library and the Multichannel Tip Wash Automated Labware Positioner or ALP as described in the text protocol. Manually load the Stacker 10s attached to the stacker carousel.In Hotels A through D, first load one box of AP96 P20 pipette tips in Room One. Then, load four 96-well V-bottom plates in Rooms Two through Five with two upper plates containing stock concentrations from the ordered library and the other two lower plates empty. Additionally, load one box of AP96 P20 pipette tips in Room Six.Load four 96-well V-bottom plates in Rooms Seven through Nine with the two upper plates containing stock concentrations of the ordered library and the two lower plates empty. Set up the deck with 300 milliliters of water in the reservoir on P3, a 300-milliliter 70%ethanol bath on P7, the Tip Loader ALP, and the Multichannel Tip Wash ALP. Using the liquid-handling robot's operating software, present AP96 P20 pipette tips from the Stacker 10 and move them to the Tip Loader ALP.Present Room Two 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.Load AP96 P20 pipette tips with the Tip Loader ALP onto the 96-channel 200-microliter head. Aspirate 90 microliters from the 300-milliliter water reservoir and dispense into the 96-well V-bottom dilution plate on P4.Repeat the step for the plate on P8.Mix the chemical library plate on P5 by repetitively aspirating and dispensing 15 microliters three times. Additionally, aspirate 10 microliters from the chemical library plate on P5 and dispense 10 microliters into the dilution plate on P4.Mix the solutions of the plate on P4 by repetitively aspirating and dispensing 50 microliters a total of three times.Once mixed, clean the AP96 P20 pipette tips by aspirating and dispensing 70 microliters of 70%ethanol from P7, then wash them in the Multichannel Tip Wash ALP by aspirating and dispensing a 110%volume of water four times. Repeat these steps for the second pair of plates on P8 and P9.Upon creating the second 96-well V-bottom dilution plate, stack the P9, P5, P8, and P4 plates in that order from bottom to top. Then, place the stack on one empty static ALP.Use either P1, P2, P6, P10, P11, P12, or P13. Repeat these steps until Room Five in Hotel A is empty, then start the process again upon reaching Room Six, moving new AP96 P20 pipette tips to the Tip Loader ALP and placing the used AP96 P20 pipette tips on an empty static ALP. Repeat the procedure until Room 9 of Hotel A is empty.In order to proceed to Hotel B, the plates and tips on the deck must be reloaded into Hotel A.Though the robot can work unsupervised, it is necessary to refill the water reservoir after each dilution cycle. If the cycle restarts with insufficient water, the robot will aspirate air and the chemicals will not be diluted. Manually refill the 300-milliliter water reservoir.The computer program can incorporate a pause detailing this message, requiring the user to hit Continue before carrying out the next step. Repeat the dilution steps for the remaining three Hotels. Manually add seeds to the media at a density of 0.1 gram per 100 milliliters.This density results in an average of 3-10 seeds per well of a 96-well plate. Manually place four 96-well flat-bottom plates in Rooms One and Two of Hotel A.Place a box of AP96 P250 pipette tips on the Tip Loader ALP, a 300-milliliter reservoir filled with the media-seed mixture on P3, and a 300-milliliter reservoir filled with 70%ethanol on P7.Using the operating software, present Rooms One and Two in Hotel A and separate the stacks of four plates. Place one plate on each of the empty static ALPs, then load AP96 P250 pipette tips on the 96-channel 200-microliter head.Aspirate 90 microliters from the 300-milliliter media-seed reservoir on P3 and dispense it into the first 96-well flat bottom plate. Repeat this process until all eight plates contain the media-seed mixture. Clean the AP96 P250 pipette tips by aspirating and dispensing 70 microliters from the 300-milliliter reservoir filled with 70%ethanol on P7.Wash the tips in the Multichannel Tip Wash ALP by aspirating and dispensing a 110%volume of water four times, then unload the tips at TL1.Finally, gather the plates by hand. Manually load a box of AP96 P250 pipette tips into Room One of Hotel A.Also, load two 96-well V-bottom dilution library plates into Rooms Two, Four, Six, and Eight. Load two 96-well flat-bottom screening plates into Rooms Three, Five, Seven, and Nine.Using the operating software, configure the deck to contain a wash reservoir of 300 milliliters of 70%ethanol at P7.The media-seed reservoir can be left on the deck at P3.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. Present the AP96 P250 pipette tip box from Hotel A and move it to the Tip Loader ALP.Next, present the 96-well V-bottom dilution library plates from Room Two of Hotel A to the deck. Place one plate on static ALP P4 and one plate on P8.Then, present the 96-well flat-bottom screening plates from Room Three of Hotel A to the deck. Place one plate on static ALP P5 and one on P9.Load AP96 P250 pipette tips with the Tip Loader ALP onto the 96-channel 200-microliter head.Mix the 96-well V-bottom dilution plate on P4 by aspirating and dispensing 50 microliters three times. Following that, aspirate 10 microliters from this plate and dispense into the 96-well flat-bottom screening plate on P5.Mix the solutions in the plate at P5 by aspirating and dispensing 50 microliters three times. Clean the AP96 P250 pipette tips with ethanol by aspirating and dispensing 70 microliters of 70%ethanol from the reservoir at P7.Then, wash the tips in the Multichannel Tip Wash ALP by aspirating and dispensing a 110%volume of water four times.Repeat these steps for the second 96-well V-bottom dilution library plate and 96-well flat-bottom screening plate. 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 the static ALPs.Then, repeat the process 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 confirmation. Supplement the wells with fewer than three seeds by addition of sterilized and vernalized seed.It is critical to ensure that the media in which the seedlings are growing does not dry out, as this will result in poor germination. Plates containing seedlings should be kept at high humidity or stored in desiccation-resistant containers. Incubate the 96-well flat-bottom screening plates for four days in an environmental chamber at 22 degrees Celsius on a 16/8 light/dark cycle in a desiccation-proof container.Visualize the 96-well flat-bottom screening plates under a dissecting microscope and record all aberrant phenotypes for further investigation. Examples of normal and aberrant phenotypes are no visible morphological abnormalities, brown root hairs, stunted root, severely stunted root, bleached, green mucilage, incomplete germination, and no germination. Visible phenotypes by percentage are displayed in a pie chart.Seedlings exhibited abnormalities in root morphology, pigmentation, root hairs, and germination. Also included is a variety of others, which include the production of green mucilage. Once mastered, this technique can greatly increase the efficiency and accuracy of large forward chemical genetic screens.While attempting this procedure, it is important to remember to sterilize and vernalize seed, refill the water reservoir during dilution steps, ensure samples are stored in desiccation-resistant containers, and that all imaging is done in flat-bottom plates. Depending on the physiological process you are interested in investigating, this initial screen can be followed up with additional assays to help determine the mode of action of each chemical. After watching this video, you should have a good understanding of how to use a liquid-handling robot to improve the efficiency and accuracy of a chemical genetic screen.

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Bioengineering

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