Dandekar & Chandler:Casein growth experiments

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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 after the first few days of passage into 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 inert gas if using for anaerobic experiments (steps 1-4)

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

For anaerobic experiments:

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)

Concentrated base for PM

for 1000 mL

20 g	Nitrilotriacetic acid (NTA-free acid)
28.9 g	MgSO4 anhydrous
6.67 g	CaCl2·2H2O
0.0185 g	(NH4)6Mo7O24·4H2O
0.198 g	FeSO4·7H2O
100 ml	Metal 44 (recipe follows)

Directions:

Dissolve NTA separately in 600 ml water and neutralize with KOH (14.6 g KOH)

Add other components, and dissolve in order given

Adjust to pH 6.8 before making to final volume of 1000 ml

- A precipitate forms when adjusting the pH from the acid side of 6.8 with KOH (need about 100 ml of 1M KOH), but eventually will redissolve with stirring

- When the pH is near 6.8, the color of the solution changes from a deep yellow to straw color

Filter sterilize and store in glass bottle wrapped with aluminum foil

Store at 4ºC for ~one year

Metal 44

for 1000 ml metal 44

2.5 g	EDTA (free acid, not sodium salt)
10.95 g	ZnSO4·7H2O
5 g	FeSO4·7H2O
1.54 g	MnSO4·H2O
0.392 g	CuSO4·5H2O
0.25 g	Co(NO3)2·6H2O
0.177 g	Na2B4O7·10H2O

Directions:

Add EDTA to 800 ml distilled water, stirring

Adjust pH to ~5.0 with 10M NaOH to dissolve EDTA

Add the other metals in the order given

- Do not add components until the previous one has completely dissolved!

Add ddH20 to final volume of 1000 ml

- Final pH should be around 2.4 and it should appear as a clear, lime green solution

Filter sterilize and store in glass bottle wrapped with aluminum foil

Store at 4ºC indefinitely

Casein growth protocols

Starting casein cultures **Updated 04/02/14**

4 days before the start of experiment

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

3 days before

Pick a single colony and inoculate into LB + 50mM MOPs buffer

Grow O/N at 37°C with shaking

2 days before

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

Grow ~ 30 hours at 37°C with shaking

• Culture should turn milky white within the first 4(ish) hours of inoculation
• Future days of passaging consistently grow to the expected density when the Day 1 culture is allowed to grow ~30 hours; Day 1 culture passaged at 24 or fewer hours does not reliably grow to optimal density even if the culture shows the milky phenotype
• If WT culture does not become milky this day or any subsequent day, you should start over -- your cultures are below the quorum threshold, and regardless of the amount of non-milky culture you passage to the next day, you will not be able to re-establish optimum cell density and experimental setups will fail or be difficult to reproduce

1 day before

After ~24 hours, into fresh 1% casein transfer **200** μL from Day 1 casein culture to start Day 2 casein culture

• Previous protocol said transfer 100 μL, but as insurance that you will always have enough cells to keep QS on, it is recommended to transfer a larger volume; we have settled on 200 μL
• Again, if cultures do not show the milky phenotype within half a day, you should go back to plates and LB overnight culture to start again

Grow O/N at 37°C with shaking

Use this Day 2 culture to set up casein experiments or inoculate into flasks to grow overnight if larger volumes of cells are needed