TITLE: Effect of supplementation of Iron and Enterobactin on C. elegans behaviour on Keio E. coli mutants (6-well plates)
AUTHORS: Saul   Moore

[DESCRIPTION]
Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their differential effects on Caenorhabditis elegans behaviour when supplemented with iron and enterobactin.


[STEPS]
SECTION: Preparing NGM agar + pouring plates
1. Prior to screening, prepare the materials needed for screening C. elegans on selected Keio E. coli mutants:

- 6-well plates (aka. 'imaging plates')
- 15 mL Falcon tubes
- 50 mL Erlenmeyer flasks
- 90 mm Petri plates (aka. 'maintenance plates')
- 150 mm Petri plates (aka. 'nursery plates')

SECTION: Preparing worms
9. Inoculate 10ml LB broth media with E. coli BW25113 (Keio background wild-type strain, used as negative control and for raising worms, no Kanamycin) in an Erlenmeyer flask for overnight culture following the protocol: 
 

SECTION: Preparing bacteria
24. Fill 2 separate Erlenmeyer flasks with 25 mL LB. Add 50μg/ml Kanamycin to one flask, and leave the other flask without Kanamycin for the BW25113 control. 
SECTION: Seeding imaging plates (6-well)
30. Remove the imaging plates from 4°C storage
SECTION: Picking worms + Hydra tracking (6-well)
42. Prior to tracking, ensure that the imaging cave air conditioning is turned on (and there has not been a power-cut) and also empty the dehumidifier waste water tray (see pre-imaging checklist)


SECTION: Preparing NGM agar + pouring plates
2. Make 1L normal Nematode Growth Media (NGM) agar, following the protocol:
 

SECTION: Preparing NGM agar + pouring plates
4. Using the Integra ViaFill, dispense 4 mL NGM agar into each well of the 6-well plates, following the protocol: 
 

SECTION: Preparing NGM agar + pouring plates
5. Leave the plates on the lab bench (with lids on) until the agar has cooled and solidified (approximately 1 hour, timing depends on humidity)
SECTION: Preparing NGM agar + pouring plates
6. Measure the weight of 3 imaging plates (with lids on) and record average plate weight on day of pouring
SECTION: Preparing NGM agar + pouring plates
7. Dry the imaging plates under a hood (or drying cabinet) until the plates lose between 3-5% of their original plate weight (with lids on)
SECTION: Preparing NGM agar + pouring plates
8. Store the imaging plates upside-down at 4°C until used for experiments
SECTION: Preparing NGM agar + pouring plates
3. Pour 15 mL NGM agar into each 60 mm maintenance plate, and 35 mL NGM agar into each 90 mm nursery plate, following the protocol for Plate pouring (dx.doi.org/10.17504/protocols.io.6bhhaj6). 
Keep the remaining agar warm in a water bath set to 65°C, for pouring into 6-well imaging plates afterwards
SECTION: Preparing worms
10. Place the inoculation in a shaking incubator at 37°C at 200 rpm and leave to grow overnight
SECTION: Preparing worms
11. Remove the BW culture from the shaking incubator and place in 4°C fridge until seeding
SECTION: Preparing worms
12. Remove the plates from storage and the BW culture from the fridge, and leave on the bench for approximately 30 minutes to acclimate to room temperature
SECTION: Preparing worms
13. Using aseptic technique, seed the 60 mm maintenance plates each with approximately 250 μL of BW25113 culture
SECTION: Adding iron and enterobactin (6-well)
35. On the day of tracking, remove the seeded imaging plates from 4°C, and dry for 30 minutes under a laminar flow hood
SECTION: Preparing worms
14. Leave under hood until dry (with lids on, timing depends on humidity)
SECTION: Preparing worms
15. Using a platinum pick, gently pick 30 adult N2 Bristol C. elegans onto each maintenance plate, and store in an incubator at 20°C
SECTION: Preparing worms
16. After 24 hours, remove the adult worms, leaving the eggs behind to hatch into L1 larvae
SECTION: Preparing worms
17. Inoculate a further 10 mL LB broth with BW25113 bacteria for overnight culture (no Kanamycin), following the protocol in   and place in a shaking incubator at 37°C, 200 rpm

SECTION: Preparing worms
18. After 24 hours, remove the culture from the incubator, and the 90 mm nursery plates from storage, and leave to acclimate on bench top for 30 minutes
SECTION: Preparing worms
19. Seed the nursery plates each with approximately 1 mL of fresh BW25113 culture. Leave under hood until dry
SECTION: Preparing worms
20. Wash the worms off the BW-seeded maintenance plates, into two 15ml Falcon tubes
SECTION: Preparing worms
21. Perform an egg prep on worms in the Falcon tubes, following the protocol: 
 

SECTION: Preparing worms
22. At around noon the next day, wash L1-arrested larvae off the empty plate and re-feed onto the BW-seeded nursery plates using a glass Pasteur pipette. Aim to dispense around 500 worms per plate. 
SECTION: Preparing bacteria
25. Remove the required Keio frozen stock plates from -80°C containing the strains for antioxidant testing. Gently remove the aluminium film and leave to partially thaw for a minute or so
 

SECTION: Preparing bacteria
26. Inoculate the Erlenmeyer flasks with the desired strains for antioxidant testing from Keio frozen stock plates, following the protocol:
 

SECTION: Preparing bacteria
28. Remove the overnight cultures from the incubator. Inoculate 2 more Erlenmeyer flasks for a second round of overnight cultures from the first, this time without Kanamycin (to avoid exposing the worms to the antibiotics), and incubate overnight at 37°C at 200 rpm.


SECTION: Seeding imaging plates (6-well)
31. Ensure that imaging plates have lost approximately 3-5% of their original weight (so that they are not too wet for imaging when seeded). Place under a hood or drying cabinet until they have.
SECTION: Seeding imaging plates (6-well)
32. Remove overnight cultures of Keio strains from 4°C storage. Using a pipette, seed 30 μL of bacterial culture into the wells of each 6-well imaging plate.


SECTION: Seeding imaging plates (6-well)
33. Place the seeded plates under a laminar flow hood to dry for 20 minutes, then place in an incubator at 25°C (no shaking) for 7 hours 40 minutes (total lawn growth time: 8 hours)
SECTION: Seeding imaging plates (6-well)
34. After 8 hours total growth time, remove the plates from the incubator and store at 4°C
SECTION: Adding iron and enterobactin (6-well)
39. Leave the plates to dry in a biosafety hood for 30 minutes. 
SECTION: Adding iron and enterobactin (6-well)
37. Using a pipette, dispense 40 μL of iron(III)chloride and iron(III)sulphate into the desired wells (on top of the lawns) of the 6-well imaging plates (for a final concentration of 1 and 4 mM iron in 4 mL agar)


SECTION: Picking worms + Hydra tracking (6-well)
43. Remove the nursery plates of worms from the 20°C incubator.
SECTION: Picking worms + Hydra tracking (6-well)
44. Using a platinum worm pick, carefully pick 10 Day1 worms onto the edge of the bacterial lawns in each well of the 6-well imaging plates, then place in incubator at 20°C until tracking (after 4 hours on food + supplements). 


SECTION: Picking worms + Hydra tracking (6-well)
45. 30 minutes prior to tracking with the Hydra rig (each run is performed every 20-30 minutes), remove 5 imaging plates at a time from the 20°C incubator and leave to acclimate in the imaging cave.
SECTION: Picking worms + Hydra tracking (6-well)
47. Place the plates under the Hydra rig and record worm behaviour on the bacterial food for 15 minutes (at the 4-hour timepoint, 25 fps, exposure: 25000 msec, blue-light stimulation)
SECTION: Picking worms + Hydra tracking (6-well)
48. After tracking, discard the plates in a biological waste bin
SECTION: Picking worms + Hydra tracking (6-well)
49. Check tracking checklist to ensure that all videos have been saved correctly: '/Volumes/behavgenom$/Documentation/Protocols/analysis/tracking-checklist-20210210.docx'
SECTION: Preparing bacteria
27. Incubate the cultures overnight at 37°C in a shaking incubator at 200 rpm.
SECTION: Adding iron and enterobactin (6-well)
36. Remove the iron(III)chloride, iron(III)sulphate and enterobactin from 4°C. 
Prepare 100 and 400 mM of iron(III)chloride and iron(III)sulphate (in H2O)
Prepare 1 mg/mL enterobactin solution (in DMSO)
SECTION: Preparing bacteria
29. After 24 hours, remove the cultures from the incubator and store at 4°C until used for experiments
SECTION: Adding iron and enterobactin (6-well)
40. Record the weight of the plates after drying (as weight at imaging).
SECTION: Preparing worms
23. Incubate at 20°C for 68 hours until the worms are Day 1 adults for the experiment
SECTION: Adding iron and enterobactin (6-well)
41. Then leave the plates on the bench (with lids on) for a further 1 hour 30 minutes (total 2 hours with the supplements added) before adding worms.
SECTION: Picking worms + Hydra tracking (6-well)
46. Wipe the underside of the lids using KIMTECH Science lint-free precision wipes (to remove any condensation that has formed)
SECTION: Adding iron and enterobactin (6-well)
38. Using a pipette, dispense 5 µL of enterobactin solution on top of the lawns of the desired wells in the 6-well imaging plates (for a final concentration of 1.25 µg/mL enterobactin in 4 mL agar)