Dandekar & Chandler:Casein growth experiments: Difference between revisions
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For anaerobic experiments: | 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 [http://www.chemglass.com/product_view.asp?pnr=CLS-4208 hungate tubes] (including lids and rubber stopper) into anaerobic glove box | 1. Pour PM into round flask, flush with argon gas for 30 min, close with rubber stopper, and place medium and 16 ml [http://www.chemglass.com/product_view.asp?pnr=CLS-4208 hungate tubes] (including lids and rubber stopper) into anaerobic glove box | ||
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:Add other components, '''and dissolve in order given''' | :Add other components, '''and dissolve in order given''' | ||
:Adjust to pH 6.8 before making to final volume of 1000 ml | :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 | ::- 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 | ::- 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''' | :Filter sterilize and store in glass bottle '''wrapped with aluminum foil''' | ||
:Store at 4ºC for ~one year. | :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== | ==Casein growth protocols== | ||
===Starting casein cultures=== | ===Starting casein cultures **Updated 04/02/14**=== | ||
''4 days before the start of experiment'' | ''4 days before the start of experiment'' | ||
:From frozen stock, streak to LB plate and grow O/N at 37°C | :From frozen stock, streak to LB plate and grow O/N at 37°C | ||
''3 days before'' | ''3 days before'' | ||
:Pick a single colony and inoculate into LB + | :Pick a single colony and inoculate into LB + 50mM MOPs buffer | ||
:Grow O/N at 37°C with shaking | :Grow O/N at 37°C with shaking | ||
''2 days before'' | ''2 days before'' | ||
:Into an 18 mm tube with 3 mL 1% casein, transfer 30 μL O/N LB culture (Day 1 casein culture | :Into an 18 mm tube with 3 mL 1% casein, transfer 30 μL O/N LB culture (Day 1 casein culture) | ||
:Grow | :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'' | ''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 | :Grow O/N at 37°C with shaking | ||
Use this Day 2 culture to set up casein experiments | |||
Use this Day 2 culture to set up casein experiments or inoculate into flasks to grow overnight if larger volumes of cells are needed |
Revision as of 13:51, 2 April 2014
Back to Protocols
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