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{{:Team:DTU-Denmark/Templates/StartPage|Experiment 4}} <div class="overviewPage"> ==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: # ''E. coli'' cultures will be grown at 37 ^oC in the incubator and # ''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) *[[Team:DTU-Denmark/Methods/Modified_DM_minimal_medium|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: # 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) # 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) # Prepare 1 L ''Nitrosomonas'' ATCC medium containing ammonia. This is needed for: #* overnight culture (10 mL) and resuspending (400 mL) x2 # 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 # Prepare 50mM ammonium chloride solution. #* Dissolve 2.4544 g NH4Cl in 50 mL DM modified minimal medium (no NH₄Cl). # 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''. # Grow'' E. coli'' top10 overnight in 10mL of DM medium + NH₄Cl prepared in step 1 at 37^oC in an erlenmeyer flask. # Take 4mL of E. coli overnight culture and add to 400mL fresh DM medium + NH₄Cl. # 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 . # Pellet down the 400mL culture at 3000g for 4 min at room temperature. # Wash with 5 mL Modified DM minimal medium and centrifuge again. # 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. # 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: # Remove one aliquot of 100mL of DM medium (without the added NH₄Cl). Repeat the following steps for each experiment and for the control: # Put the experimental flask on the hot plate with magnet stirrer and stabilize the temperature at 37^oC (26^oC for ''Nitrosomonas''). # 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. # Use 2mL of the first sample to measure the OD. # Take a 2mL sample at 5 min and at 10 min for a baseline. # 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. # Then take another sample immediately after the spike. # Continue taking samples every 5 min until t=30 min. # Take samples every 10 min until t=60 min. At the end of the experiment: # Take an extra 2mL sample to measure the OD. # Measure the temperature. To finish gathering data: # Make the colorimetric measurements for: #* ammonium for each of the samples collected for the AMO experiment by using the protocol [[Team:DTU-Denmark/Methods/Ammonium_colorimetric_measurements#Ammonium_colorimetric_measurements|Ammonium measurement]]. #* nitrite for each of the samples collected for the HAO experiment by using the protocol [[Team:DTU-Denmark/Methods/Nitrite_colorimetric_measurements|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). <div style="clear: both;"></div> </div> {{:Team:DTU-Denmark/Templates/EndPage}}

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