Team:DTU-Denmark/Methods/Determining concentration of nitrogen compounds/Experiment 4

From 2013.igem.org

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 @ 26C
• 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 2 experimental flasks:

1. E. coli, to which spikes of NO₂^- will be added.
2. P. aeruginosa, to which spikes of NO₂^- will be added.

In addition to the continuous gas measurements, we take samples to measure the concentration of ammonia, nitrite and nitrate at the beginning and end of each spike.

The response time of the cells to spikes of NO₂^- is expected to be on the order of minutes, and we will run the experiment until the solution has reached saturation with NO₂^-.

EQUIPMENT NEEDED

• 1 400 mL bottle with a lid that the electrodes can be fastened air tightly to
• 1 magnetic stirrer
• 1 NO probe for NO measuring
• 1 N₂O probe for N₂O measuring
• Syringes with long needles and small needles
• Syringe filters with pore size 0.2μm
• Stopwatch
• Modified DM minimal medium
• E. coli overnight culture
• P. aeruginosa overnight culture
• Flat bottom centrifuge tubes
• 2mL Eppendorf tubes
• 10mL, 1mL, 200μL pipettes with tips.
• MilliQ water

EXPERIMENTAL PROCEDURE

First,

1. Prepare DM minimal medium with ammonium chloride as a nitrogen source. Add 0.745 g NH₄Cl to 1L of prepared DM medium.
2. Start overnight cultures.
3. Prepare test solutions for ammonium, nitrite and nitrate kits.
4. Label eppendorf tubes and test tubes for colorimetric samples.
5. Grow E. coli top10 overnight/ P. aeruginosa in 10mL of DM medium + NH₄Cl prepared in step 1 at 37^oC.
6. Take 4mL of E. coli/P. aeruginosa overnight culture and add to 200mL fresh DM medium + NH₄Cl.
7. Grow the cells at 37^oC in 200 RPM until OD=0.35 (about 3 hours; about 5 hours for P. aeruginosa).
8. Pellet down the 200mL culture, 3000g for 4 min (Cool down the centrifuge if it is needed for 30 min at 4^oC).
9. Wash with 5 ml cold Modified DM minimal medium and centrifuge again.
10. Pour off the supernatant and resuspend the cell pellet in 200mL Modified DM Minimal medium in a centrifuge tube and pour samples together if they were made in more than one tube.
11. Measure OD of the 200 mL cell suspension and add Modified DM minimal medium until OD=0.3 (note the exact value).
12. Pure the 200 mL of the OD=0.3 suspension to a bottle. This is the experimental flask.
13. For the control: Remove one aliquot of 200mL of DM medium (without the added NH₄Cl).
14. Make the anaerobic experiment by saturating with pure N₂ following the Sparging with N₂ method , for 5 min the nitrite stock solution and 10 min the cell suspensions and control.
15. Start the experiment by adjusting the NO and N₂O electrodes in one flask at a time.
16. Put the flask on the magnetic stirrer and start with 270 rpm at 37^oC.
17. Remove 2 mL as the first sample t=0 colorimetric analysis.
18. Add 0.5 mL of 50mM nitrite stock solution to 100 ml of the cell suspension.
19. Watch the concentrations of NO and N₂O, and continue adding nitrite when they are not changing (about 10 min).
20. Repeat steps 18-20 until the solution exceeds the sensitivity of the sensor (1mM).
21. Make the colorimetric measurements for nitrite, nitrate and ammonium for each of the samples collected by using the Nitrite measurement, Nitrate measurement and Ammonium measurement, respectively in order to finish gathering data.

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