Spormann Lab:Protocols:ReactorMedium

In Development. 2013-12-18 Now outdated

MOPS Buffered Medium

0. Rinse Medium Bottle with de-ionized or MQ water

1. In large 2L rinsed mixing beaker add

1. 40 ml of 50x Salt Solution
2. 1860 ml of MQ de-ionized water (avoid water with high Fe content).
3. 40 ml of 50x sodium MOPS, pH 7.45 (1 M stock, filter-sterilized). Check the pH of the medium.
4. 2ml Trace element solution (1000×)
5. 2ml Se/Wo solution (1000×)
6. 0.5ml Resazurin (1 mg/ mL)

3. Add the solution and a 2 inch stir bar to the 2L GL45/GL25-side port influent vessel equipped with three port cap blue GL45 cap.

4. Add 1860 ml of MQ de-ionized water to the vessel

5. Loosely connect GL25 cap so that pressure may escape during autoclaving process.

6. Add GL45 - 3 port cap with luer-equipped gas input.

7. Autoclave on liquid cycle for 45 minutes.

8. Let liquid cool for 10 minutes in Autoclave.

9. Immediately transfer the hot liquid to back room under stream (>0.1Bar) of N2/CO2

10. Let cool for 15 minutes while gas stream is passing.

11. Transfer the vessel to ice-water bath to cool, before adding additional components.

12. When cool to the touch, Add:

1. 2ml of 1 M L-Cysteine solution to final concentration of 1mM
2. 0.4ml of 1M Sodium Sulfide Solution to a final concentration of 0.2mM

13. Wait for 15-30 minutes. allowing 0.1 Bar N2 gas to flow over the Medium. The color of the solution should turn to bright pink and eventually become clear, indicating that residual oxygen has been removed.

14. When reduced and cool.

1. Add 2 ml of 1000x Vitamin Solution
2. Depending on protocol you may also want to add yeast extract 0.05 % w/v as a supplement, particularly on reactor startup.

15. Close gas port to the reactor vessel and tighten GL25 top cap. Spray Leur adapter with 70% ethanol and place a sterile plug in until ready to attach to reactor. Store, covered in aluminum foil and protect from light.

Components:

50x Salt Solution (In Stock 900 ml 2012-12-18) g/L

1. 50 NaCl
2. 20.3 MgCl2 x 6H2O
3. 13.4 NH4Cl
4. 26.1 KCl
5. 0.75 CaCl2 x 2H2O
6. 10.2 KH2PO4

MOPS Buffer (pH 7.45) Mixture of 1M Free-Acid and 1M NaMOPS until desired pH is reached. (In Stock 900 ml 2012-12-18)

Se-Wo Solution

1. NaOH 0.5 g/L
2. Na2SeO3 x 5H2O 6 mg/L
3. Na2WO4 x 2H2O 8 mg/L

Trace Elements Solution (E) (In Stock 900 ml 2012-12-18)

g/L

1. 12.8 nitrotriacetic acid
2. 1.35 FeCL3x6H20
3. 0.1 MnCl2*4H20
4. 0.024 CoCl2*6H20
5. 0.1 CaCl2*2H20
6. 0.1 ZnCl2
7. 0.025 CuCl2*2H20
8. 0.01 H3BO3
9. 0.024 Na2MoO4*2H20
10. 1 NaCl
11. 0.12 NiCl2*6H20
12. 0.026 NaSeO3*5h20

Vitamin Solution

B12

Precipitation Tests

1ml 50x Salt Solution, 1ml 1M MOPS were prepared in 50ml batches.

1. Nothing
2. 0.25ml Se-Wo + 0.25 ml 1000x Trace Elements (E)
3. 0.25ml Se-Wo + 0.25 ml 1000x SL10
4. 0.25ml Se-Wo + 0.25 ml 1000x SL12
5. Each were autoclaved for 25 minutes