Dandekar & Chandler:Pa/Bc competition setup

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(Coculture competition protocol)
Line 5: Line 5:
Streak strains of interest from frozen stock
Streak strains of interest from frozen stock
-
::- One parent Pa, one QS mutant Pa, one competitive Bc
+
::*One parent Pa, one QS mutant Pa, one competitive Bc
-
::- Streak onto DRY plates to ensure single colonies for inoculation
+
::*Streak onto DRY plates to ensure single colonies for inoculation
'''Morning before:'''
'''Morning before:'''
-
::- Using single colonies from overnight plates, inoculate 3 ml LB + 50mM MOPS in 16mm tubes
+
::*Using single colonies from overnight plates, inoculate 3 ml LB + 50mM MOPS in 16mm tubes
-
::- Grow ~6-8hrs
+
::*Grow ~6-8hrs
'''Evening before:'''
'''Evening before:'''
-
::- From morning culture, make serial dilutions to ensure there is < 0.5 OD tube for the next morning
+
::*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
+
:::- 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
+
:::- 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
+
:::- If dilutions are made at 5pm, 9 am is 16 hours of growth; check tubes immediately upon arriving to lab
'''Morning of:'''
'''Morning of:'''
-
::- Check dilution tubes 5-7; hopefully one will be ≤ OD 0.5
+
::*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
+
::*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 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
 +
|}

Revision as of 19:05, 20 August 2013

Back to Dandekar & Chandler: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
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