TITLE: Keio Screen
AUTHORS: Saul Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol for screening </span><span style = "font-style:italic;">Caenorhabditis elegans</span><span> behavioural response to bacterial gene-deletion mutants from the 'Keio Collection', to identify behaviour-modifying mutations for follow-up analyses.</span></div></div>

[STEPS]
?. [Seeding bacterial lawns (96WP)]
Ensure that the imaging plates have lost approximately 3-5% of their original weight. Place under a hood (or drying cabinet) until this is the case. (Friday, -4 days)
?. [Seeding bacterial lawns (96WP)]
Remove overnight cultures for 'Old Lawn' plates that were prepared in Step 24, and seed 10μl bacterial culture into each well of 4 imaging plates using the Integra ViaFlo.(Friday, -4 days)
?. [Inoculating from Keio stock plates (96WP)]
Fill the wells of two 96-well culture plates (Nunc™ 96-Well Polystyrene Round Bottom Microwell Plates, Non-Treated) with 200μL LB broth media and 50μg/ml Kanamycin.(Wednesday, -6 days)
?. [Preparing maintenance plates]
Prior to the experiment, prepare 35 NGM agar-filled 60mm Petri plates (worm maintenance plates). To make 500mL NGM agar, follow the steps in the protocol for Making normal NGM for imaging plates (Cabreiro Lab) and pour 15ml NGM agar into each plate:(Friday, -11 days)
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?. [Preparing NGM imaging plates (96WP)]
Prepare 500ml NGM agar by following the steps in the protocol Making normal NGM for imaging plates (Cabreiro Lab):(Wednesday, -6 days)
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?. [Seeding bacterial lawns (96WP)]
Remove imaging plates from 4°C storage and record the average weight of three of the plates.(Friday, -4 days)
?. [Dispensing worms using COPAS (96WP)]
Remove 2 'New Lawn' and 2 'Old Lawn' seeded plates from 4°C, and dry for 30 minutes under a hood. Once dry, leave for a further 30 minutes to acclimate to room temperature.(Monday, -1 day)
?. [Tracking using Hydra rigs]
Remove the imaging plates (with L4-dispensed worms) from the 20°C incubator and leave to acclimate in the imaging cave for 30 minutes prior to tracking (+24 hours and +25 hours on food, ie. after dispensing with COPAS in Step 41).(On Tuesday, Tracking Day 1)
?. [Preparing maintenance plates]
Seed the remaining 25 maintenance plates with fresh BW25113 culture, and leave under hood until dry.(Wednesday, -6 days)
?. [Preparing maintenance plates]
Inoculate 10ml LB broth media with BW25113 bacteria (used as negative control and for raising worms) for overnight culture, following the protocol, Inoculating a Liquid Bacterial Culture, and place in a shaking incubator at 37°C at 200 rpm:(Friday, -11 days)
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?. [Preparing maintenance plates]
Seed the 10 maintenance plates with 120μL of BW25113 culture(Saturday, -10 days)
?. [Preparing maintenance plates]
Using a platinum pick, gently pick 7 adult N2 Bristol C. elegans onto each of the 10 seeded maintenance plates, and store in an incubator at 20°C.(Monday, -8 days)
?. [Preparing maintenance plates]
Remove the adult worms after 24 hours, leaving the eggs behind to hatch into L1 larvae that will be raised on BW25113.(Tuesday, -7 days)
?. [Preparing maintenance plates]
Inoculate 10ml LB broth media with BW25113 bacteria for overnight culture and place in a shaking incubator at 37°C, 200 rpm.(Tuesday, -7 days)
?. [Preparing maintenance plates]
Wash the worms off the 10 BW-seeded maintenance plates prepared in Step 4, into two 15ml Falcon tubes.(Friday, -4 days)
?. [Preparing maintenance plates]
The next day at about midday, wash the L1 larvae off the empty plate and re-feed onto the 25 newly seeded maintenance plates using a glass Pasteur pipette. Aim to dispense around 500 worms per plate. (Saturday, -3 days)
?. [Preparing maintenance plates]
Perform an egg prep on worms in the Falcon tubes, by following the protocol for Egg Prep for Bleach Synchronization (Cabreiro Lab):(Friday, -4 days)
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?. [Preparing NGM imaging plates (96WP)]
Using the Integra ViaFill, dispense 200μL of NGM agar into each well of 8 imaging plates (Whatman Square Well Flat Bottom UNIPLATE), following the protocol Dispensing agar into multiwell plates:(Wednesday, -6 days)
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?. [Preparing NGM imaging plates (96WP)]
Leave the plates on the lab bench (with lids on) until the agar is dry.(Wednesday, -6 days)
?. [Preparing maintenance plates]
Label 12 plates for harvesting L4 worms, and 13 plates for harvesting Day 1 adult worms, and incubate at 20°C.(Saturday, -3 days)
?. [Preparing NGM imaging plates (96WP)]
Measure the weight of 3 plates (with lids on) and record average plate weight on day of pouring.(Wednesday, -6 days)
?. [Preparing NGM imaging plates (96WP)]
Store the plates upside-down at 4°C (plates can be stored for up to 1 week prior to use).(Wednesday, -6 days)
?. [Inoculating from Keio stock plates (96WP)]
Inoculate the 96-well culture plates from the Keio stock plate, following the protocol for Growing overnight bacterial culture in 96WP (Cabreiro Lab):(Wednesday, -6 days)
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?. [Inoculating from Keio stock plates (96WP)]
Remove Keio frozen library stock plate from -80°C, gently remove the aluminium film, and then leave the wells to defrost for a few minutes.(Wednesday, -6 days)
?. [Inoculating from Keio stock plates (96WP)]
Incubate thee cultures overnight at 37°C (no shaking).(Wednesday, -6 days)
?. [Preparing NGM imaging plates (96WP)]
Dry the plates under a hood (or drying cabinet) until the plates lose between 3-5% of their original plate weight (with lids on)(Wednesday, -6 days)
?. [Inoculating from Keio stock plates (96WP)]
Repeat Steps 18 - 21 to inoculate from Keio frozen stocks again for the 'New Lawn' imaging plates (ie. freshly seeded the day before imaging).(Friday, -4 days)
?. [Inoculating from Keio stock plates (96WP)]
Repeat Step 21 to incubate overnight at 37°C (no shaking). These cultures ('twice removed' from frozen stocks) will be used for seeding the 'Old Lawn' imaging plates (ie. seeded 3 days prior to imaging and refrigerated). (Thursday, -5 days)
?. [Inoculating from Keio stock plates (96WP)]
Remove the overnight cultures from the incubator, and inoculate the newly prepared culture plates from yesterday's overnight culture plate using a replicator, following the above protocol for Growing overnight bacterial culture in 96WP (Cabreiro Lab). (Thursday, -5 days)
?. [Inoculating from Keio stock plates (96WP)]
Repeat Step 18 to prepare 2 more overnight culture plates. (Thursday, -5 days)
?. [Inoculating from Keio stock plates (96WP)]
Repeat Steps 22 - 24 for the second round of overnight cultures for the 'New Lawn' plates.(Saturday, -3 days)
?. [Seeding bacterial lawns (96WP)]
Place seeded plates under a 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).(Friday, -4 days)
?. [Seeding bacterial lawns (96WP)]
After 8 hours, remove the plates from the incubator and store overnight at 4°C.(Friday, -4 days)
?. [Seeding bacterial lawns (96WP)]
Remove overnight cultures for 'New Lawn' plates that were prepared in Step 26, and seed 10μl bacterial culture into each well of 4 imaging plates using the Integra ViaFlo. (Sunday, -2 days)
?. [Seeding bacterial lawns (96WP)]
Repeat Steps 30 - 31 for the 'New Lawn' plates.(Sunday, -2 days)
?. [Tracking using Hydra rigs]
Record Day 1-dispensed worm behaviour on thee bacterial food for 15 minutes at the +24 hour timepoint.(On Wednesday, Tracking Day 2)
?. [Dispensing worms using COPAS (96WP)]
Remove 12 maintenance plates with L4-stage worms (prepared in Step 11) and wash the worms off the plates into two 15ml Falcon tubes using approximately 10ml PBS 'A' buffer (sterile). (Monday, -1 day)
?. [Dispensing worms using COPAS (96WP)]
Fill up the tubes to 15ml with PBA 'A' and centrifuge at 1000rpm for 2 minutes.(Monday, -1 day)
?. [Dispensing worms using COPAS (96WP)]
Remove the supernatant using a Pasteur pipette. (Monday, -1 day)
?. [Dispensing worms using COPAS (96WP)]
Repeat Steps 36 - 37 four more times to thoroughly rinse off any BW25113 bacteria. (Monday, -1 day)
?. [Dispensing worms using COPAS (96WP)]
Re-suspend the worms and divide them equally into two 50ml Falcon tubes (for the COPAS), and fill them both up to approximately 40ml with PBS 'A'. (Monday, -1 day)
?. [Dispensing worms using COPAS (96WP)]
Dispense 3 L4-stage worms into each well of the 4 imaging plates (2 'New', 2 'Old') using the COPAS following the protocol COPAS wormsorter:(Monday, -1 day)
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?. [Dispensing worms using COPAS (96WP)]
Turn on the COPAS machine by following Steps 1 - 16 of protocol COPAS wormsorter to prepare and clean the machine for dispensing L4-stage worms:(Monday, -1 day)
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?. [Dispensing worms using COPAS (96WP)]
Leave the plates to dry under a hood for 30 minutes, then incubate at 20°C overnight.(Monday, -1 day)
?. [Dispensing worms using COPAS (96WP)]
Repeat Steps 34 - 40 for the 13 remaining maintenance plates with Day 1 adult worms to prepare them for COPAS. (Tuesday, Tracking Day 1)
?. [Dispensing worms using COPAS (96WP)]
Dispense 3 Day 1 adult worms into each well of the 4 imaging plates (2 'New', 2 'Old') using the COPAS following the protocol COPAS wormsorter:(Tuesday, Tracking Day 1)
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?. [Dispensing worms using COPAS (96WP)]
Leave the plates to dry under a hood for 30 minutes before placing them in the imaging cave to acclimate for a further 30 minutes prior to tracking using the Hydra imaging rig.(Tuesday, Tracking Day 1)
?. [Tracking using Hydra rigs]
Record L4-dispensed worm behaviour on the bacterial food for 15 minutes at the +24 hour and +25 hour timepoints (25 fps, exposure: 25000 msec, with blue-light stimulation).(On Tuesday, Tracking Day 1)
?. [Tracking using Hydra rigs]
Record Day 1-dispensed worm behaviour on thee bacterial food for 15 minutes at the +1, +3, and +5 hour timepoints.(On Tuesday, Tracking Day 1)
?. [Tracking using Hydra rigs]
Remove Day 1-dispensed imaging plates from the 20°C incubator 30 minutes prior to tracking +24 hour timepoint, and leave to acclimate in the imaging cave.(On Wednesday, Tracking Day 2)
?. [Tracking using Hydra rigs]
After imaging the +5 hour timepoint for Day 1-dispensed worms, store the Day 1-dispensed imaging plates in the 20°C incubator overnight.(On Tuesday, Tracking Day 1)
?. [Tracking using Hydra rigs]
After tracking, discard the plates in a biological waste bin.
?. [Preparing maintenance plates]
Dry 10 maintenance plates for approximately 2 hours (depending on humidity).(Saturday, -10 days)
?. [Preparing maintenance plates]
Leave to dry on the bench top for about half an hour, and then store upside-down in an incubator at 25°C. (Saturday, -10 days)
?. [Inoculating from Keio stock plates (96WP)]
Wet some tissue with MilliQ water, and wrap the culture plates in the tissue.NB: The tissue provides humidity that aids growth, while the presence of Kanamycin should prevent contamination.(Wednesday, -6 days)