Competition Assays (Mixed-Population Samples Vs. Ancestor)
The following protocol is for the standard competition assays, in which for a given generation each of the 12 populations is competed against the reciprocally-marked ancestor with three-fold replication.
- REL606 (Ara- ancestor), REL607 (Ara+ ancestor), and 12 mixed-population samples.
- ~80 TA plates, ~1.5 l of DM25, ~200 ml of LB.
Label 14 flasks by strain. Add 9.9 ml of LB to each. Ancestral strains can be "looped" into appropriate flasks. Evolved populations must be allowed to thaw slightly, then use pipetter to remove 0.1 ml of each into appropriate flasks.
Label 72 flasks, 18 for each baseline strain and 3 for each evolved strain. Add 9.9 ml DM25 to each. Dilute each LB culture by 10-4 (via one dt) into 18 (baseline) or 3 (evolved) corresponding flasks.
Label 36 flasks, 3 for each evolved strain. Add 9.9 ml DM25 to each. Label 36 TA plates, also noting t=0. Pair-up yesterday's preconditioning flasks for the evolved populations and ancestral strains possessing the opposite Ara marker. Use each preconditioned strain only once. For each pair, mix 0.05 ml of each competitor into the corresponding fresh DM25 flask, then immediately sample at 2 x 103 (via one dt, then spread 0.05 ml) onto correct TA plate.
Label 36 TA plates, also noting t=1. Sample each competition culture at 2 x 105 (via two dts, then spread 0.05 ml) onto correct TA plate. Count t=0 plates.
Count t=1 plates.