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.
