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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 trafficking^1,2,3,4,5. 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 70^{9,11,12,13,14,15,16}. 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 biology^{17,18,19,20,21}. 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's key outcomes in 4 steps.

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. Please click here to view a larger version of this figure.

Protokół

1. Creating a Dilution Library

1. 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).
2. 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.
3. Load, by hand, the Stacker 10'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 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 plates containing stock concentrations of the ordered library and the two lower plates empty (Figure 5, Stacker).
4. 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).
5. 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's operating software; see Table of Materials.
6. 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).
7. Load AP96 P20 Pipette Tips with the Tip Loader ALP onto the 96-Channel 200 µL Head. Aspirate 90 µL 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.
8. Mix the chemical library plate on P5 by repetitively aspirating and dispensing 15 µL three times. Additionally, aspirate 10 µL from the chemical library plate on P5 and dispense 10 µL into the dilution plate on P4.
9. Mix the solutions of the plate on P4 by repetitively aspirating and dispensing 50 µL a total of three times. Once mixed, clean the AP96 P20 Pipette Tips by aspirating and dispensing 70 µL 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.
10. 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.
11. 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.
12. 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.
13. 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 'continue', before carrying out the next step.
14. 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.

2. Adding Media-seed Mixture to Screening Plates

1. Make ½ 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.
2. 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 °C from 24 hours to 7 days for vernilization. The Arabidopsis Biological Resource Center describes additional methods of sterilization, vernilization, and growth²².
3. 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.
4. 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.
5. 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 µL Head.
6. Aspirate 90 µL 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.
7. Clean the AP96 P250 Pipette Tips by aspirating and dispensing 70 µL 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.

3. Adding Small Molecules to Screening Plates

1. 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.
2. 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.
3. Present the AP96 P250 Pipette Tip Box from Hotel A and move it to the Tip Loader ALP.
4. 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.
5. Load AP96 P250 Pipette Tips with the Tip Loader ALP onto the 96-Channel 200 µL Head.
6. Mix the 96-Well V-Bottom Dilution Plate on P4 by aspirating and dispensing 50 µL three times. Following that, aspirate 10 µL from this plate and dispense into the 96-Well Flat-Bottom Screening Plate on P5.
7. Mix the solutions in the plate at P5 by aspirating and dispensing 50 µL three times. Clean the AP96 P250 Pipette Tips with ethanol by aspirating and dispensing 70 µL 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.
8. 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).
9. 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.
10. 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.

4. Incubation and Visualization of Screening Plates

1. Incubate the 96-well Flat-Bottom Screening Plates for four days in an environmental chamber at 22 °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.

Wyniki

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...

Dyskusje

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 date^9,13,23. The use of a liquid handling robot enabled more efficient dilution libra...

Materiały

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