Day 0
- Plate strains for mating on agar, grow overnight.
- Donor – E. coli on selective LB. Grow 37°C.
- Helper – E. coli CC118 λpir / pEVS104 [MJM534] on LB-Kan50. Grow 37°C.
- Recipient - typically V. fischeri on LBS. Grow 25-28°C.
Day 1
- Grow overnight liquid cultures of the strains with antibiotics if necessary.
Day 2
- Combine 100 μl of each E. coli culture only in a 1.5-ml microfuge tube (see controls below).
- Pellet cells by centrifugation (1 min at 8,000 x g).
- Add 100 μl of each V. fischeri culture to the E. coli pellet.
- Pellet cells by centrifugation (1 min at 8,000 x g); resuspend in 10 μl fresh LBS.
- Spot this onto a fresh, thick LBS plate.
- Incubate for ~16 h at 25-28°C.
Day 3
- Scrape the mating mix off the plate and suspend in 750 μl of LBS. In the case of V. fischeri, spot should be yellowish.
- Plate on selective media and incubate at room temperature (or cooler, e.g., 15°C).
- This cooler temp enriches for Vibrio over the E. coli donor.
- For "frequent" events like introduction of a stable plasmid, plate dilutions and/or streak purify from the spot.
- Colonies should appear in 1-2 days at room temperature, or 3-4 days at 15°C. Streak-purify transconjugants.
A generic tri-parental mating includes an E. coli strain with pEVS104 as the conjugal helper plasmid, a second E. coli strain with an oriT-containing plasmid to be mobilized, and a recipient Vibrio. For quadraparental mating (ex. with mini-Tn7) use 200 µl of the Vibrio recipient.
Controls: Same as conjugation tube but excluding one of the constituent strains (no donor, no recipient, and no helper controls).
Plate freshness: Fresh LBS plates seem to help a lot. If possible, use them the same day you pour them; just make sure they’re not "sloppy wet." If you want to compare mating efficiencies of different constructs, be careful to use similar plates or multiple spots on the same plate.