Experiment 4

Procedure

In this aerobic experiment, we add increasing concentrations of ammonium NH₄⁺ to transformed E. coli cells growing aerobically, in order to test whether our AMO transformant is converting ammonium to hydroxylamine (NH₂OH) . The same protocol is used to measure whether our HAO transformant is converting hydroxylamine to nitrite NO₂^-.

The bottle is placed to a magnetic stirrer on 270 rpm and 37^oC.

The AMO experiment will need the following:

• AMO E. coli transformant (duplicate)
• Control: Untransformed E. coli.
• Control: Nitrosomonas at 26 ^oC
• Control: Abiotic

We expect the AMO transformant to use ammonium more quickly than the untransformed control, and Nitrosomonas to also use ammonia.

The HAO experiment will need the following:

• HAO E. coli transformant (duplicate)
• Control: Untransformed E. coli.
• Control: Nitrosomonas at 26 ^oC
• Control: Abiotic

We expect the HAO transformant and Nitrosomonas to consume hydroxylamine and produce nitrite, and the untransformed E. coli will not.

There will be 10 experimental flasks:

1. E. coli cultures will be grown at 37 ^oC in the incubator and
2. Nitrosomonas Europaea at 26 ^oC

EQUIPMENT NEEDED

• 4 erlyenmeyer flasks for growing cultures
• 1 hot plate and magnet stirrer
• Syringe filters with pore size 0.2μm
• Stopwatch.
• Ammonium Chloride (NH₄Cl)
• Modified DM minimal medium
• E. coli overnight culture
• Nitrosomonas overnight culture
• LB-broth medium
• Flat bottom centrifuge tubes
• 50 mM ammonium chloride stock solution
• 2mL Eppendorf tubes
• Colorimetric test kits for ammonium
• Rack for the samples
• Glass tubes for the colorimetric tests
• 10mL, 1mL, 200μL pipettes with tips
• Single use plastic cuvettes
• MilliQ water

EXPERIMENTAL PROCEDURE

First, prepare the solutions that will be used:

1. Prepare at least 2L DM minimal medium with ammonium chloride as a nitrogen source. Add 0.745 g NH₄Cl to 1L of prepared DM medium. This is needed for:
   • growing the AMO transformant (10 mL overnight culture + 400mL to resuspend)
   • growing the HAO transformant (10 mL overnight culture + 400mL to resuspend)
   • growing the E. coli control for both experiments (10 mL overnight culture + 400mL to resuspend x2)
2. Prepare at least 1L DM modified minimal medium (with no ammonium chloride). This is needed for:
   • washing the E. coli cultures (5mL x 6 experiments)
   • resuspending the E. coli cultures for the experiments (107.5 mL x6)
   • media for the abiotic control experiments (100mL x2)
3. Prepare 1 L Nitrosomonas ATCC medium containing ammonia. This is needed for:
   • overnight culture (10 mL) and resuspending (400 mL) x2
4. Prepare at least 500 mL Nitrosomonas ATCC medium with no ammonia source. This is needed for:
   • washing (5mL) x2
   • resuspending for the experiment (100mL) x2
5. Prepare 50mM ammonium chloride solution.
   • Dissolve 2.4544 g NH4Cl in 50 mL DM modified minimal medium (no NH₄Cl).
6. Prepare 80mM hydroxylamine solution.
   • Dissolve 0.260 g hydroxylamine in 100 mL DM modified minimal medium (no NH₄Cl).

For the AMO and HAO experiments, repeat the following for both the transformed and untransformed E. coli, and for N. europaea.

1. Grow E. coli top10 overnight in 10mL of DM medium + NH₄Cl prepared in step 1 at 37^oC in an erlenmeyer flask.
2. Take 4mL of E. coli overnight culture and add to 400mL fresh DM medium + NH₄Cl.
3. Grow the E. coli at 36.6^oC in 210 RPM until OD=0.35 (about 5 hours). Grow the N. europaea at 26^oC .
4. Pellet down the 400mL culture at 3000g for 4 min at room temperature.
5. Wash with 5 mL Modified DM minimal medium and centrifuge again.
6. Pour off the supernatant and resuspend the cell pellet. Pour samples together if they were made in more than one centrifuge tube. The OD should be around 0.3.
7. Remove one aliquot of 107.5 mL of the OD=0.3 suspension to a flask and keep at 37^oC, but don't let it sit around for many hours. This is the experimental flask.

For the abiotic control:

1. Remove one aliquot of 100mL of DM medium (without the added NH₄Cl).

Repeat the following steps for each experiment and for the control:

1. Put the experimental flask on the hot plate with magnet stirrer and stabilize the temperature at 37^oC (26^oC for Nitrosomonas).
2. Remove 4 mL as the first sample t=0 colorimetric analysis. For the abiotic control, take samples only at the start and end of the experiment.
3. Use 2mL of the first sample to measure the OD.
4. Take a 2mL sample at 5 min and at 10 min for a baseline.
5. After the min 10 sample, quickly spike with
   • 1.7 mL of 50mM ammonium stock solution. Then the ammonium concentration in 100mL is 0.85mM (equivalent to the Km of AMO) = 11.9mg/L Nitrogen.
   • 50uL of 80mM hydroxylamine solution. Then the hydroxylamine concentration in 100 mL is 0.04mM (the Km of HAO) = 0.56 mg/L Nitrogen.
6. Then take another sample immediately after the spike.
7. Continue taking samples every 5 min until t=30 min.
8. Take samples every 10 min until t=60 min.

At the end of the experiment:

1. Take an extra 2mL sample to measure the OD.
2. Measure the temperature.

To finish gathering data:

1. Make the colorimetric measurements for:
   • ammonium for each of the samples collected for the AMO experiment by using the protocol Ammonium measurement.
   • nitrite for each of the samples collected for the HAO experiment by using the protocol Nitrite measurement

If none of the the added ammonia is converted to hydroxylamine, then the last sample will contain 11.9 mg N-Ammonia/L (the test kit measures between 0 and 70 mg/L approx).

If all the added hydroxylamine is converted to nitrite, then the last sample will contain 0.56 mg N-Nitrite/L (the test kit measures between 0 and 1 mg/L approx).