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)
• If experiment is to be conducted in SCFM, reinoculate low OD cells from LB to SCFM to allow them to adapt to SCFM growth conditions

- While there are no noticeable inconsistencies in PA01/PA01ΔLasR SCFM runs inoculated with cells grown in LB, it’s a real possibility that future parent-mutant strains will perform differently;

- Better to keep experiment “pure” to avoid inconsistent performance that may be the result of switching growth medium

Setting up:

• Make sure there are enough surface dry LB agar + antibiotic plates

- Use trimethoprim to select for Pa

- Use 3x abx to select for Bc

• Label plates using two replicates of each plate, allowing 2 rows of spots for each biological replicate in the experiment
• Fill a multichannel pipette trough with experimental medium
• Use multichannel 20-200 ul multichannel pipette to fill 96-well plate wells with 180 ul of experimental medium
• Label 96-well plate with each culture and replicate
• Using 18mm test tubes with 3 ml experimental medium, label and prepare duplicate tubes for each competition scenario and controls:

Controls

Parent Pa A	Parent Pa B
Mutant Pa A	Mutant Pa B
Burkholderia A	Burkholderia B

• Set up WT Parent Pa vs Bc competition ratios:

10:1 A	10:1 B
1:10 A	1:10 B
1:1 A	1:1 B

• Set up Mutant Pa vs Bc competition ratios:

10:1 A	10:1 B
1:10 A	1:10 B
1:1 A	1:1 B