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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 | |||
|} |
Latest revision as of 15:56, 14 February 2014
Back to Protocols
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