Dandekar & Chandler:Protocols

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Media

• M9 Salts
• Selective Media
• Mueller Hinton II (Cation Adjusted)
• LB

General Protocols

• MIC Protocol
• Screen for Bactobolin Resistant Mutants
• AHL Extraction
• Pyocyanin Extraction
• AHL Signal Measuring
• Electroporation with pseudomonas or burkholderia
• Pseudomonas Mating
• Colony PCR for non-E. coli strains
• PCR "sewing"

Specific Pseudomonas Projects

• Casein growth experiments

Casein growth FYIs

• 1% casein medium is somewhat opaque, but this is not why OD cannot be used as a measure of growth
• As pseudomonas elastase/proteases break down casein, the turbidity in the tube increases as the bits of degraded protein accumulate

-- WT PAO1 will turn the tube milk white and completely opaque after ~4 hours in fresh medium

-- A LasR mutant should NOT show the milky tube phenotype! If it does, you have contamination

if you want to keep the casein solution you have made, be sure to keep a negative control to rule out contamination there

• To enumerate growth, colony counts should be performed

Casein media recipes

1% casein medium

- 1% Casein sodium salt from bovine milk [from Sigma]

- 1x PM medium or 1x M9

Stir at medium high speed until casein is dissolved (can take anywhere from 1-3 hours depending on how awesome your stir plate is)

Filter sterilize

-- Note that filter surface area is the same size in the 250 ml and 500 ml filter sterilization units; because the filters will clog, it's better to aliquot into 250 ml or smaller bottles

PM Medium

for 1000 mL "requires flushing with argon

800 ml ddH2O 25 ml 0.5M Na2HPO4 25 ml 0.5M KH2PO4 10 ml 10% (NH4)2SO4 10 ml concentrated base (recipe follows)

- Bring volume to 1000 ml and pH to 6.8

- Autoclave for 45 min

1. Pour PM into round flask, flush with argon gas for 30 min, close with rubber stopper, and place medium and 16 ml hungate tubes (including lids and rubber stopper) into anaerobic glove box 2. Pour 10 ml into hungate tubes, and close with rubber stopper and lid 3. Remove tubes from glove box and autoclave 4. Add carbon sources from sterile stock solutions (e.g., acetate and succinate)

Casein growth protocols

Starting casein cultures

Day 1

From frozen stock, streak to LB plate and grow O/N at 37°C

Day 2

Pick a single colony and inoculate into LB + 5mM MOPs buffer

Grow O/N at 37°C with shaking

Day 3

Into an 18mm tube with 3 mL 1% casein, transfer 30 μL O/N LB culture

Grow O/N at 37°C with shaking

Day 4

Into fresh 1% casein, transfer 100 μL from Day 1 casein culture

Co-Culture Projects

• Pa/Bc Competition