Dandekar & Chandler:Pa/Bc competition setup

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Pseudomonas:Burkholderia co-culture competition protocol

Two days before:

Streak strains of interest from frozen stock

- One parent Pa, one QS mutant Pa, one competitive Bc

- Streak onto DRY plates to ensure single colonies for inoculation

Morning before:

- Using single colonies from overnight plates, inoculate 3 ml LB + 50mM MOPS in 16mm tubes

- Grow ~6-8hrs

Evening before:

- From morning culture, make serial dilutions to ensure there is < 0.5 OD tube for the next morning

For consistency between experiments, it’s important to make sure that your starting culture hasn’t turned on QS; Once cells have entered stationary phase, and become QS-on, they will continue to be QS-on even with back diluting

Using 3 ml LB, set up 7 tubes per culture; make 1st tube a 10X dilution, and continue 10x dilutions through the series

- If dilutions are made at 5pm, 9 am is 16 hours of growth; check tubes immediately upon arriving to lab

Morning of:

- Check dilution tubes 5-7; hopefully one will be ≤ OD 0.5

- Cells above OD 0.6 may already be QS-on; discard those tubes

- If cells are in the 0.1-0.2 OD range, let them continue to grow while setting up 96-well plate and antibiotic agar plates are set up (check on them ~30 min later)