| Materials | Details |
|---|---|
| Plastic cups | Webstaraunt store: 619PI9 |
| Nalgene 1000 ml Filtration unit | Fisher Scientific 158-0200 |
| Transfer pipets | Fisherbrand: 13711-9AM |
| LBS agar | . |
| Glass plating beads |
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Day 1 - Pipet 90 µl LBS into 1.5 ml microfuge tube.
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Scrape approximately 15 μl of library frozen stock into a separate 1.5 ml microfuge tube. This should thaw within a few minutes.
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Pipet 10 µl thawed frozen stock into the LBS. Vortex for 3 s then shake at 25 °C for 1 h. Note: if there are 80+ squid scale up, but be sure to keep the ratio at 1:10.
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Collect juvenile squid in plastic bowls with fresh FSIO (check and correct salinity prior to filtering).
- Record the number of squid from each clutch used in the experiment.
- Transfer the squid to ambient light once hatching is complete; wait until after colonization to return to the dark.
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Prepare the juvenile squid as follows:
- APO bowl – 40ml fresh FSIO – 5 squid
- SYM bowl – 40ml fresh FSIO – 40 squid
- Duplicate SYM bowl depending on number of juveniles collected. Note. At most 40 squid per bowl but may use less.
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Begin 3 h inoculation - Add 40 μl of culture into into each SYM bowl. Dispense inoculum throughout bowl, then mix water vigorously using a transfer pipet to evenly distribute bacteria throughout the bowl (remember to mix through the center of the bowl).
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Set a timer for 3 h after inoculation.
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Plate Inoculum - Perform a 1:100 dilution of contents in SYM bowl by transferring 10 µl to a tube containing 990 µl LBS.
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Plate 50 µl of resulting dilution onto LBS agar plates containing 5 sterile glass beads.
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Stop inoculation - Transfer juvenile squid to 40 ml fresh FSIO and place in the dark in the squid facility overnight
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Day 2 - Before lights out (11:00 am), transfer squid to 40 ml fresh FSIO.
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Pool the vent samples from the SYM bowls into a single bowl; mix well and transfer 40 ml to a 50 ml conical tube. Centrifuge 10,000 x g for 10 min.
- Discard supernatant
- Repeat until all vent has be pelleted.
- Store at -20C with day1_date-vent#1.
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Return the squid to squid facility overnight
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Day 3 - Before lights out (11:00 am), measure and record luminescence for all APO juveniles (6 s standard integration).
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Measure and record data luminescence for 2-4 squid from each SYM bowl. If not uniformly colonized then record more animals.
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Freeze the squid in separate 1.5 ml microfuge tubes at -80 °C.
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Pool the vent samples from the SYM bowls into a single bowl; mix well and transfer 40 ml to a 50 ml conical tube. Centrifuge 10,000 x g for 10 min.
- Discard supernatant
- Repeat until all vent has be pelleted.
- Store at -20C with day1_date-vent#2.
Twenty microliters from the input mutant library stock was thawed, diluted 10-fold in LBS, vortexed for 3 s, and incubated at 25 °C for 1 h with aeration on a cell culture rotator. After 1 h, 40 μL of the diluted library was added to a plastic cup containing 40 mL filter-sterilized Instant Ocean (FSIO) and ≤40 E. scolopes squid hatchlings. FSIO containing squid was inoculated with ∼2 × 105 cfu/mL of the library for 3 h. Squid were then transferred to 40 mL un-inoculated FSIO for an additional 45 h (water was changed at 24 h postinoculation), at which point they were killed and surface sterilized by storage at −80 °C. Individual squid were then homogenized in 700 μL FSIO, and 50 μL of each homogenate was plated on LBS agar using standard methods (Ref. 10). Bacterial colonies from each plate were scraped with a sterile cell scraper, collected, and subsequently diluted 1:10 in FSIO. Fifty microliters of the dilution, for n = 250 squid platings, were collected in a 50 mL conical tube and stored at −80 °C before DNA isolation and INSeq analysis.