Nov 21, 2024
FoldSeek screen
- James Marshall1,
- Eleanor Warren1,
- John Bergqvist1
- 1MRC Laboratory of Medical Sciences, Imperial College London
- Behavioural Genomics
Protocol Citation: James Marshall, Eleanor Warren, John Bergqvist 2024. FoldSeek screen. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqd563vk5/v1
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: November 12, 2024
Last Modified: November 21, 2024
Protocol Integer ID: 111966
Abstract
This protocol outlines the screening procedure of a library of transformed E. coli expressing proteins with insecticidal potential. The two outputs from this procedure is 1) the OD600 as a measurement of how much C. elegans have consumed the transformed E. coli, and 2) recording worms under bluelight stimulation to capture variations in motility.
Materials
100 mm Petri dishes
Round-bottom Nunclon 96-well plates
2 mL deep 96-well plates
Breathable film cover for 96-well plates
Lysogeny Broth medium
Nematode Growth Medium
M9 medium
S-medium
Kanamycin
Nystatin
IPTG
Bleach solution
MilliQ water
C. elegans (N2)
E. coli (OP50)
Day 1 - Preparing worms
Day 1 - Preparing worms
30m
30m
Egg laying
Pick 20 adult worms onto six 100 mm plates seeded with 1.5 mL of OP50. Leave on plates for 24:00:00 h .
30m
Day 2 - Removing adult worms & making S-medium
Day 2 - Removing adult worms & making S-medium
2h 20m
2h 20m
Remove adult worms from the 100 mm plates if sufficient eggs have been laid
20m
Check if S-medium is available.
If not, make S-medium.
Note
1 L Pre-autoclave S-medium (S-basal)
NaCl 5.85 g 4th floor
K2HPO4 1 g 4th floor
KH2PO4 6 g 4th floor
MilliQ water 1 L 4th floor
Note
1 L Post-autoclave S-medium
*Kanamycin, Nystatin and IPTG do not last long and so should only be added to smaller aliquotes upon use.
Cholesterol (5 mg/mL in ethanol) 1 mL 4th floor
1 M potassium citrate 6 10 mL 4th floor
trace metals solution 10 mL 4th floor
1 M CaCl2 3 mL 4th floor
1 M MgSO4 3 mL 4th floor
--------------------------------------------------------------------------
1000x Kanamycin* 1 mL 4th & 6th floor
5000x Nystatin* 200 µL 4th floor
1 M IPTG* 100 µL 4th & 6th floor
If needed, make more 1 M IPTG by mixing 2.38g IPTG in 10 mL sterile water, followed by filtering using a 0.22 µm filter.
2h
Day 4 - Beginning of Bacterial Growth
Day 4 - Beginning of Bacterial Growth
30m
30m
Grow FoldSeek Library of plates
Fill the four pre-made FoldSeek transformed E. coli plates with 100 µL of LB broth+Kanamycin. Cover plates with breathable film cover.
Leave overnight at 37 °C and shaking at 200 rpm.
Note
Make LB+Kanamycin by pipetting 500 µL of 1000x Kanamycin into 500 mL LB.
30m
Day 5 - Inducing bacteria & bleaching worms
Day 5 - Inducing bacteria & bleaching worms
5h 30m
5h 30m
Dilute overnight cultures 1 in 100
Dilute 5 µL of overnight cultures in 450 µL LB broth+Kanamycin in 2 mL deep-well plates (LB+Kan can be filled by ViaFill using programme '16 DEEP WELL').
30m
Grow diluted cultures to OD600 0.4 - 0.8
Incubate the plates at 37 °C 230 rpm for about 03:00:00 h until they have reached an OD600 between 0.4 - 0.8.
3h
Induce bacterial cultures with IPTG at OD600 0.4 - 0.8
Make a 10x solution of 1 M IPTG (1000x) in LB+Kanamycin.
When cultures have grown to an OD600 of 0.4 - 0.8, use ViaFill (programme '16 DEEP WELL') to dispense 50 µL of LB+Kan+IPTG in each well. Rinse ViaFill tubes with heated MilliQ water.
Leave overnight at 37 °C and 250 rpm.
Note
4x 96-well plate:
50 µL x 384 wells = 19.2 mL
The ViaFill requires about 5 mL for priming. As such, to ensure there is enough dispensed without risking bubbles, make up a solution of 40 mL.
10x IPTG in LB+Kanamycin (1/100 dilution):
400 µL of 1 M IPTG (1000x IPTG) in 40 mL of LB+Kanamycin.
30m
Bleach worms and place in diapause
Need around 50-100,000 L1s. To be used on Day 6, so leave spinning overnight.
1h 30m
Day 6. Add worms to bacteria & measure
Day 6. Add worms to bacteria & measure
3h 15m
3h 15m
Complete S-meduim
Use a 500 mL measuring cylinder aliquote 400 mL of S-medium into a new autoclaved glass container. Add the following components to complete the S-medium:
Note
400 mL complete S-medium:
1000x Kanamycin 400 µL 4th & 6th floor
5000x Nystatin 80 µL 4th floor
1 M IPTG 40 µL 4th & 6th floor
Kanamycin, Nystatin and IPTG available on 4th floor 'Toxic' -20°C freezer in box labelled 'Spare antibiotics/antifungals' or in yellow boxes in a -20°C freezer on 6th floor.
Make more 1 M IPTG by mixing 2.38g IPTG in 10 mL sterile water, followed by filtering using a 0.22 µm filter.
10m
Pellet & re-suspend bacteria in S-medium
Pellet plates for 00:10:00 min at 500x g. Flick out the LB and replace the medium with 500 µL of S-medium using ViaFill.
Note
1630x rpm for centrifuge 5810R to get 500x g.
30m
Centrifuge again and refill with S-medium
Fill the wells with 300 µL of S-medium using ViaFill. Rinse the ViaFill tubes with MilliQ water to ensure no clogging occurs.
5m
Resuspend & make triplicates plates
Using a multichannel pipette, resuspend the cultures thoroughly.
Pipette 80 µL into three separate Nunclon round-bottom 96-well plates.
1h
Add worms to the plates
Add 20 µL of L1 worms to each well (between 50 - 100 worms).
30m
Measure OD
Measure OD600 of the plates using a plate reader (with 'James_OD_600' protocol).
File naming format: FS21_1_JB1_Day1
- FS: FoldSeek
- 21: plate 2.1
- 1: plate replicate 1 out of 3 of FS plate 2.1
- JB1: experiment repeat for initals
- Day1: day of the measurement out of 14.
30m
Record plates using Hydras
Plates recorded without lids using only one hydra for the entire screen.
Script: python3 run_Short_bluelight.py -r 02 -f ToxAss_2024_MM_DD_JB1_d1_FS21_1
- MM : month for when the first recording was made
- DD : day for when the first recording was made
- JB1: experiment repeat for intials
- d1: day of the measurement out of 14
- FS21: FoldSeek plate number 2.1
- 1: plate replicate 1 out of 3 of FS plate 2.1
The script runs for 30s. When the blue light has stopped, the recording is done. Take out the plate and put in the new one. Make sure to click 'clear' under 'configurations' before starting to record the new plate.
30m
Store plates
Place plates in a box with a damp paper towel and store at 20 °C
Day 9 to Day 19 - Measuring OD600 and motility
Day 9 to Day 19 - Measuring OD600 and motility
1h
1h
Measure OD600 using the plate reader
Use the protocol 'James_OD_600'.
30m
Record plates using Hydras
Run the script 'run_Short_bluelight.py' to record the plates under bluelight.
Note
14 days in total for measurements (Day 6 - Day 19). Measurements done on Day 6 and from Day 9 to Day 19 of Experiment Days. If the first measurement starts on Monday, then the second day of measurement is on Thursday.
Exp. Day (Measurement day) Day of week
1. Day 6 (1) Mon
2. Day 9 (4) Thurs
3. Day 10 (5) Fri
4. Day 11 (6) Sat
5. Day 12 (7) Sun
6. Day 13 (8) Mon
7. Day 14 (9) Tues
8. Day 15 (10) Wed
9. Day 16 (11) Thurs
10. Day 17 (12) Fri
11. Day 18 (13) Sat
12. Day 19 (14) Sun
30m