Oct 28, 2020
Syngenta divergent strain screen protocol
- 1Imperial College London
- Behavioural Genomics
Protocol Citation: Ida Barlow, Adam Mcdermott-Rouse, Luigi Feriani 2020. Syngenta divergent strain screen protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bn5zmg76
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 28, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 43929
Abstract
Protocol for screening the 12 divergent C. elegans strains with 100 Syngenta pesticide drugs at 3 concentrations and imaging under baseline and bluelight conditions using the Hydra (Loopbio) imaging rigs. The twelve strains were imaged over two days of tracking and on each day of tracking six strains were imaged across all drugs and all concentrations with 3 replicates for all conditions. This protocol should be repeated at least 3 times.
Guidelines
Careful planning of how drugs to be arranged in plates and the number of strains is required before undertaking screening experiments. Using a google calendar to pre-plan timings and days is advised in order to efficiently manage the workload
Materials
Equipment
VIAFLO
NAME
96 channel pipette
TYPE
Integra
BRAND
VIAFLO 96
SKU
LINK
Equipment
VIAFILL
NAME
reagent dispenser
TYPE
Integra
BRAND
VIAFILL
SKU
LINK
Before start
96 well plates containing the drug library at all doses need to be randomised by column using the OpenTrons robot to create 3 stock plates for each library plate.
Pick L4 worms for bleaching (-9 days from first day of tracking)
Pick L4 worms for bleaching (-9 days from first day of tracking)
1h
1h
Pick 10 x L4s for 6 strains onto 3 x 90mm plates (pre-seeded with OP50) for each strain
Pick L4 worms for bleaching and pour 96WPs (-7 days)
Pick L4 worms for bleaching and pour 96WPs (-7 days)
4h
4h
Pick 10 x L4s for 6 strains onto 3 x 90mm plates (pre-seeded with OP50) for each strain
Prepare 2 x 1.5L low peptone NGM and autoclave
Once agar has cooled to around 65 °C , add the salts and dispense agar into 140 x square well 96 well plates using VIAFILL dispenser. Dispense 200 µL per well. Once cooled, store agar side up in an airtight container at 4 °C
Dry plates, bleach worms (-5 days)
Dry plates, bleach worms (-5 days)
4h
4h
Dry 20 x 150mm plates in cabinet dryer (setting 2) or 3 hours
Seed 20 x 150mm plates with OP50 and leave to dry overnight at room temperature
Bleach worms prepared for day 1 of tracking
Protocol
NAME
Bleach synchronisation of C. elegansCREATED BY
Ida Barlow
Wash hermaphrodites off plate with several ml of M9 solution and transfer to 15ml falcon tube (Fisher Scientific-Falcon 352096)
Fill falcon tube up to 15ml with M9 solution
Centrifuge for 2 minutes at 1500 rpm (RCF:210, ascending 9; descending 7) – program 1
Program 1 retains the worms as pellets and the bacteria is suspended as the supernatant
The descending is slow as the worm pellet is lose at this stage which we don't want to break
Remove supernatant using a plastic Pasteur pipette taking care not to disturb pellet
Leave atleast 0.5ml M9 to avoid disturbing the pellet
Fill the tube with M9 upto 15ml
Spin program 1
Repeat steps 4-6
On final wash remove as much supernatant as possible and add M9 upto 4ml
Add 4ml 2X Bleach solution (From here onwards try to work as quickly as possible to avoid over-exposure of the worms to the bleach)
USE FRESHLY PREPARED BLEACH EVERYTIME
Note
2X Bleach solution:
5% Sodium hypochlorite solution - 4ml
Sterile water - 3.5 ml
1M NaOH solution - 2.5 ml
TOTAL - 10 ml
Vortex on maximum setting for 4 min (no more as this will damage the eggs)
Makesure the vortex forms
After vortexing, top up the tube with M9 till 15ml
Centrifuge for 2 mins at 2500rpm (RCF:590, ascending 9; descending 7) – program 2
(Always check the program on the centrifuge before using it)
Remove supernatant by pouring into waste bottle – pellet should be compact and yellow in colour at bottom of falcon, but be careful not to lose
Add 15ml M9
Centrifuge at program 2
Repeat steps 12-14 four more times
The number of washes is crucial here as we need to get rid of all the bleach
After final wash add 15ml M9 and store eggs/larvae in the falcon on the rotator that is constantly spinning at 20oC, until feeding
Note
L1 arrested larvae can be starved for up to 5 days before refeeding
Centrifuge larvae on program 2 to pellet
Remove supernatant with plastic Pasteur pipette
The pellet is lose here so makesure not to disturb it
Add 15ml M9, spin to wash
On final wash leave 0.5ml M9 in falcon
Resuspend the pellet by gently tapping the tube/flicking it
Place droplet containing larvae onto seeded plate and allow to grow to desired developmental state (ie. 2 days for L4s, 2.5 days for young adults)
Use glass pipette to place the droplet onto seeded plate, avoid using plastic pipette as larvae will stick to it
Note
Development times at 20oC:
- 2 days for L4s
- 2.5 days for young adults
Note:
- If you feed larvae within 12hrs of bleaching then they develop faster than the longer arrested ones
- It is a good practice to bleach in two tubes in parallel
- If you drop the tube at any point of the process, makesure to transfer the contents into a new tube as the dropped tube may get cracked resulting in loss of worms during centrifugation/vortexing
- Any unused larvae can be topped up with M9 and stored spinning in the rotator to be re-used
- Use clean autoclaved rubber bulbs for the refeeding everytime to avoid contamination
- Put the used bulb in the box labelled 'Used Teets'
Table of Development times for different temperatures
Dry plates, bleach worms and refeed L1 (-3 days)
Dry plates, bleach worms and refeed L1 (-3 days)
4h
4h
Dry 20 x 150mm plates in cabinet dryer (setting 2) or 3 hours
Seed 20 x 150mm plates with OP50 and leave to dry overnight at room temperature
Bleach worms prepared for day 2 of tracking
Protocol
NAME
Bleach synchronisation of C. elegansCREATED BY
Ida Barlow
Wash hermaphrodites off plate with several ml of M9 solution and transfer to 15ml falcon tube (Fisher Scientific-Falcon 352096)
Fill falcon tube up to 15ml with M9 solution
Centrifuge for 2 minutes at 1500 rpm (RCF:210, ascending 9; descending 7) – program 1
Program 1 retains the worms as pellets and the bacteria is suspended as the supernatant
The descending is slow as the worm pellet is lose at this stage which we don't want to break
Remove supernatant using a plastic Pasteur pipette taking care not to disturb pellet
Leave atleast 0.5ml M9 to avoid disturbing the pellet
Fill the tube with M9 upto 15ml
Spin program 1
Repeat steps 4-6
On final wash remove as much supernatant as possible and add M9 upto 4ml
Add 4ml 2X Bleach solution (From here onwards try to work as quickly as possible to avoid over-exposure of the worms to the bleach)
USE FRESHLY PREPARED BLEACH EVERYTIME
Note
2X Bleach solution:
5% Sodium hypochlorite solution - 4ml
Sterile water - 3.5 ml
1M NaOH solution - 2.5 ml
TOTAL - 10 ml
Vortex on maximum setting for 4 min (no more as this will damage the eggs)
Makesure the vortex forms
After vortexing, top up the tube with M9 till 15ml
Centrifuge for 2 mins at 2500rpm (RCF:590, ascending 9; descending 7) – program 2
(Always check the program on the centrifuge before using it)
Remove supernatant by pouring into waste bottle – pellet should be compact and yellow in colour at bottom of falcon, but be careful not to lose
Add 15ml M9
Centrifuge at program 2
Repeat steps 12-14 four more times
The number of washes is crucial here as we need to get rid of all the bleach
After final wash add 15ml M9 and store eggs/larvae in the falcon on the rotator that is constantly spinning at 20oC, until feeding
Note
L1 arrested larvae can be starved for up to 5 days before refeeding
Centrifuge larvae on program 2 to pellet
Remove supernatant with plastic Pasteur pipette
The pellet is lose here so makesure not to disturb it
Add 15ml M9, spin to wash
On final wash leave 0.5ml M9 in falcon
Resuspend the pellet by gently tapping the tube/flicking it
Place droplet containing larvae onto seeded plate and allow to grow to desired developmental state (ie. 2 days for L4s, 2.5 days for young adults)
Use glass pipette to place the droplet onto seeded plate, avoid using plastic pipette as larvae will stick to it
Note
Development times at 20oC:
- 2 days for L4s
- 2.5 days for young adults
Note:
- If you feed larvae within 12hrs of bleaching then they develop faster than the longer arrested ones
- It is a good practice to bleach in two tubes in parallel
- If you drop the tube at any point of the process, makesure to transfer the contents into a new tube as the dropped tube may get cracked resulting in loss of worms during centrifugation/vortexing
- Any unused larvae can be topped up with M9 and stored spinning in the rotator to be re-used
- Use clean autoclaved rubber bulbs for the refeeding everytime to avoid contamination
- Put the used bulb in the box labelled 'Used Teets'
Table of Development times for different temperatures
At 17:00, spin L1s for day 1 of tracking at 2500rpm. Remove supernatent and using glass pipette, drop 4 small droplets around the edges of the plate (off food) onto 3 x 150mm plates per strain.
Allow to grow at 20 °C
Dry 96 well plates (-2 days)
Dry 96 well plates (-2 days)
3h
3h
Take 65-70 poured 96 well plates from the cold room, and weigh three random plates without their lids
Place in cabinet dryer (setting 1.5-2) and allow to dry for 2-3 hours with lids off
Weigh 3 random plates and verify that at least 3-5% reduction in weight
leave overnight at room temperature with lid
Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)
Dispense drugs onto imaging plates using VIAFLO (-1 days from day 1 of tracking)
4h
4h
Pre-label imaging plates (square well) with the imaging run and drug plate information, so that every plate on a single day of imaging has a unique plate id, for example L01_s01_01 where:
L01-04 - library plate number (out of 4 library plates)
sh01-03 - shuffle number (out of the 3 shuffled stock plates)
01-13 - imaging run number for that day
Remove the shuffled library plates (stock) plates from the-20 °C freezer, allow to thaw at room temperature and spin to collect contents at bottom of wells
Pre-label the appropriate (11) skirted 96 well plates with library plate IDs (L01-L04_sh01-sh03) to make up diluted drug plates
Dispense 19.6 µL water into each well of the prelabeled dilution plates using multichannel pipette and reagent reservoir
Using VIAFLO (hedgehog) dispenser on BG_STOCK custom program, premix drug in drug library plates in slot A, and then transfer 1.4 µL drug in DMSO to the dilution plates prefilled with water in slot B. Repeat for all drug library plates
Note
Double check the dispense volumes before making up the diluted plates
Z-heights have been configured in this program to prevent pipette and plate crashes
Using VIAFILL (octopus) dispenser, dispense 5 µL water onto 5 x predried imaging plates
Note
5 plates at a time to prevent the agar absorbing all the liquid before the drug is dispensed into the water droplet
Using VIAFLO in custom program BC_AGAR, with correct drug library plate in slot B, transfer 3 µL of diluted drug and water mixture onto the correctly labelled imaging plate in slot A.
Repeat until all imaging plates have had drug dispensed onto them.
Note
Z-heights have been configured in this program to prevent pipette tips from piercing the agar
Prepare 1:10 dilution of OP50 in M9 in a small bottle:
5 mL OP50
45 mL M9
Using VIAFILL dispenser, seed all the imaging plates with 5 µL per well
Place lids on each plate and leave drugged and seeded imaging plates overnight at room temperatue in the dark (with box ontop)
Refeed L1s for day 2 of tracking (-1 days for day 1 of tracking)
Refeed L1s for day 2 of tracking (-1 days for day 1 of tracking)
At 17:00, spin L1s for day 2 of tracking at 2500rpm. Remove supernatent and using glass pipette, drop 4 small droplets around the edges of the plate (off food) onto 3 x 150mm plates per strain.
Allow to grow at 20 °C
Day 1 imaging
Day 1 imaging
Wash worms off 150mm paltes with M9 buffer using pasteur pipette into 15ml falcons
Spin at 1500rpm for 2 minutes to pellet the worms
Remove supernatenet and fill with M9
Repeat steps 28-29
After final wash, fill falcon with M9 and transfer contents from 15ml falcon to 2 x 50ml and fill up 30ml with M9
Use COPAS wormsorter to dispense 3 worms per well (pure, no double) into 5 imaging plates at a time. Use pre-made YYYYMMDD_wormsorter.csv to plan and determine which strains to dispense into each plate.
Note
Each strain will sequentially be dispensed into 10.5 imaging plates.
Allow liquid to dry off by placing imaging plates in 20 °C incubator with lid off for 30 minutes, then replace lids and keep in 20oC incubator
Expose worms to drug for 4 hours in total as calculated from the middle wormsorter time and allow worms to acclimate for 30 minutes in the cave prior to imaging
Note
Example:
wormsorter start time 10 :00
wormsorter end time 11:00
middle wormsorter time 10:30
cave time 12:00
Imaging start time 12:30
Imaging on hydra using protocol script (5 mins prestim; 6 mins bluelight with 60 sec OFF, [10sec ON, 90sec OFF] x 3 times; 5 mins postsim)
Prepare imaging plates for day 2 of imaging (on same day as day 1 of imaging)
Prepare imaging plates for day 2 of imaging (on same day as day 1 of imaging)
Take 65-70 poured 96 well plates from the cold room, and weigh three random plates without their lids
Place in cabinet dryer (setting 1.5-2) and allow to dry for 2-3 hours with lids off
Weigh 3 random plates and verify that at least 3-5% reduction in weight
leave overnight at room temperature with lid
Perpare drug imaging plates for day 2 of imaging (+1 from day 1 of imaging)
Perpare drug imaging plates for day 2 of imaging (+1 from day 1 of imaging)
Repeat steps 16-25 for the other 6 strains
Day 2 imaging
Day 2 imaging
Repeat steps 27-35