Team:DTU-Denmark/Experiment2

From 2013.igem.org

(Difference between revisions)
(→Methods)
(→Introduction)
 
(66 intermediate revisions not shown)
Line 1: Line 1:
-
{{:Team:DTU-Denmark/Templates/StartPage|Experiment 2}}
+
{{:Team:DTU-Denmark/Templates/StartPage|Anaerobic production of nitrous oxide from nitrite}}
__TOC__
__TOC__
-
== Description ==
+
== Introduction ==
-
We grew ''E. coli'' and ''P.aeroginosa''anaerobically in the presence of varying concentrations of nitrite and measured the amount of nitrous oxide produced.
+
In this anaerobic experiment we characterize how readily nitrite is converted to nitric oxide (NO), and subsequently to nitrous oxide (N<sub>2</sub>O) by adding nitrite  (NO<sub>2</sub><sup>-</sup> ) in a series of spikes to:
 +
* an untransformed strain of ''E. coli''
 +
* an untransformed strain of ''P. aeruginosa''
 +
* Mutant 2: a strain of ''E. coli'' transformed with the Nir gene from ''P. aeruginosa''
-
== Methods ==
+
[[File:dtu-Mutant2-extra_ws.png|right]]
 +
We also monitor nitrite, ammonia and nitrate concentrations before each spike.  The concentrations of nitrous oxide is measured continually and the ammonia, nitrite and nitrate concentrations are measured at a series of time points around the spike.  For some experiments, we have data for the concentration of nitric oxide as well, however, the sensor frequently gave unreliable results and was replaced during the summer.  While we were waiting for the new one to arrive, we were not able to take NO measurements.
-
Experiment 2: Measure production of N<sub>2</sub>O from Nitrite NO<sub>2</sub><sup>-</sup> anaerobically
+
==Methods==
-
+
-
Characterization of the behavior of ''E. coli'' (untransformed cells) and ''P. aeruginosa'' while growing in the presence of NO2- in an anaerobic reaction chamber by measuring the conversion of nitrite (NO<sub>2</sub><sup>-</sup>) to nitric oxide (NO), nitrous oxide (N<sub>2</sub>O), and ammonia (NH<sub>3</sub>).
+
The experimental procedure follows [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_2|Protocol for Experiment 2]].
-
In this anaerobic experiment we add nitrite in a series of spikes to a native strain of ''E. coli'' and to a native strain of ''P. aeruginosa'' in order to test how readily nitrite is converted to nitric oxide, and subsequently to nitrous oxide. We also monitor nitrite, ammonia and nitrate concentrations before each spike.  The concentrations of both gasses are measured continually, and the ammonia, nitrite and nitrate concentrations will be measured after nitrite is spiked.
+
For the calibration of the N<sub>2</sub>O and NO electrodes we follow the [[Team:DTU-Denmark/Methods/Calibrating_Electrodes#Calibration_of_NO_and_N2O_electrodes.2Fprobes|calibration protocol]].
-
The anaerobic experiment is performed in a sealed bottle, where two polarographic electrodes that will measure nitric oxide and nitrous oxide are carefully attached to the lid. The electrodes are connected to a sensor to measure the different concentrations. The bottle is placed to a magnetic stirrer on 270 rpm and 37<sub>o</sub>C.
+
== Results and Discussion ==
-
There is one control flask, as well, containing only DM medium which will be spiked with  NO<sub>2</sub><sup>-</sup> (as will be done for the experimental flasks).  
+
In the following diagrams, the y-axis shows mg of Nitrogen (as nitrite, nitrous oxide or other N-source) per litre vs time.  Vertical lines indicate the time at which we spiked with nitrite. All experiments were performed anaerobically.
-
Additionally, we will measure the response of only anaerobic E. coli biomass without any NO2- added, and the same for anaerobic P. aeruginosa.  This will be done in the experimental flasks prior to spiking with  NO2-..
+
=== Control: Untransformed ''E. coli'' -- Round 1 ===
-
There will be 2 experimental flasks:
+
The first round of the experiment was performed on [[Team:DTU-Denmark/Notebook/8_August_2013#Lab_115| August 8<sup>th</sup>]] and [[Team:DTU-Denmark/Notebook/9_August_2013#Lab_115| August 9<sup>th</sup>]].
-
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 will take samples to measure the concentration of ammonia, nitrite and nitrate at the beginning and end of each spike.  
+
It appears that we observed a response from the cells after the third spike. However, this could also be due to fluctuations in the sensor. 
 +
[[File:DTU-Experiment-2-results.png|600px]]
-
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 N2O.
+
=== Control: Untransformed ''E. coli'' -- Round 2 ===
-
EQUIPMENT NEEDED
+
We repeated the experiment to try to reproduce the response that we saw in round 1. The experiment was performed on [[Team:DTU-Denmark/Notebook/22_August_2013#lab_115| August 22<sup>th</sup>]]. We were not able to see a response in either replicate that we did this round.  At the end of the second replicate, the sensor gave a jump in reading, but due to the quick drop off, it is most likely that this is due to fluctuation in the sensor and is not a reliable reading.
-
• 1 100 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 (Appendix 6)
+
-
• E. coli overnight culture
+
-
  • P. aeruginosa overnight culture
+
-
• Flat bottom centrifuge tubes
+
-
• 2mL Eppendorf tubes
+
-
• 10mL, 1mL, 200μL pipettes with tips.
+
-
• MilliQ water.
+
-
All preparations with cells will be done on ice so that the cells don’t grow.
+
-
EXPERIMENTAL PROCEDURE
+
[[File:Dtu Experiment 2 round 2 --sample A.png|600px]]
 +
[[File:Dtu Experiment 2 round 2 -- sample B.png|600px]]
-
First,
+
=== Untransformed ''P. aeruginosa'' ===
-
1. Prepare DM minimal medium with ammonium chloride as a nitrogen source.  Add 0.745 g NH3Cl 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.
+
-
Repeat the following for both E. coli and P. aeruginosa cultures:
+
We also did not see a response for the P. aeruginosa, which we would expect.
-
5. Grow E. coli top10 overnight in 10mL of DM medium + NH3Cl prepared in step 1 at 37◦C.
+
-
6. Take 4mL of E. coli overnight culture and add to 400mL fresh DM medium + NH3Cl.
+
-
7. Grow the cells at 36.6◦C in 210 RPM until OD=0.35 (about 3 hours; about 5 hours for P. aeruginosa).
+
-
8. Cool down the centrifuge for 30 min at 4◦C.
+
-
9. Pellet down the 400mL culture, 3000g for 4 min at 4◦C.
+
-
10. Wash with 5 ml cold Modified DM minimal medium and centrifuge again.
+
-
11. Pour off the supernatant and resuspend the cell pellet in 100mL Modified DM Minimal medium in each centrifuge tube and pour samples together if they were made in more than one tube.
+
-
12. Measure OD of the 100 mL cell suspension and add cold Modified DM minimal medium until OD=0.3 (note the exact value).
+
-
13. Remove one aliquot of 100 mL of the OD=0.3 suspension to a bottle and keep on ice.  This is the experimental flask.
+
-
For the control:
+
The experiment was performed on [[Team:DTU-Denmark/Notebook/7_August_2013#Lab_115|August 7<sup>th</sup>]].
-
14. Remove one aliquot of 100mL of DM medium (without the added NH3Cl).
+
 
 +
[[File:Dtu pseudo exp.png|600px]]
-
Repeat the following steps for each experiment and for the control:
+
=== Nir Transformed ''E. coli'' ===
-
15. Make the anaerobic experiment by saturating with pure N2 following the method for injecting N2 (Appendix 1), for 3 min the nitrite stock solution and 5 min the cell suspensions and controls.
+
-
16. Start the experiment by adjusting the NO and N2O electrodes in one flask at a time.
+
-
17. Put the flask on the magnetic stirrer and start with 250 rpm at 36.6◦C.
+
-
18. Remove 2 mL as the first sample t=0 colorimetric analysis.
+
-
19. Add 0.5 mL of 50mM nitrite stock solution to 100 ml of the cell suspension. Then the nitrate concentration is about 0.25mM; 3.5 mg N / L.
+
-
20. Watch the concentrations of NO and N2O, and continue when they are not changing (about 10 min).
+
-
21. Repeat steps 15-17 until the solution is exceeds the sensitivity of the sensor (1mM).
+
-
To finish gathering data:
+
Finally, we measured both N<sub>2</sub>O and NO, but did not see an increase in N<sub>2</sub>O. The NO sensor did not stabilize over the course of the experiment, and these results are not realistic.  Additionally, the N<sub>2</sub>O sensor was calibrated correctly, but when the experimental readings were converted to mg nitrogen, the values were negative.  Since this is not realistic, it is likely that the readings are not accurate and that possibly the sensor should be replaced.  Unfortunately, we did not have enough time to repeat the experiment with a new sensor.
-
22. Make the colorimetric measurements for nitrite, nitrate and ammonium for each of the samples collected by using the appendices 3 and 4.
+
-
== Results ==
+
The experiment was done on the 19th of September and the procedure is described in [[Team:DTU-Denmark/Notebook/19_September_2013|our notebook for September 19<sup>th</sup>]].
-
[[File:DTU-Experiment-2-results.png|600px]]
+
[[File:Dtu-nir.png|600px]]
 +
 
 +
<div style="clear: both;"></div>
 +
 
 +
== Conclusions ==
-
Mg of Nitrogen (as nitrite or nitrous oxide) per litre measured for varying concentrations of Nitrite added at t=0.  All experiments were performed anaerobically.   
+
Despite multiple replicates over multiple experiments, we were not able to reliably detect nitrous oxide being produced in any strain as a result of the addition of nitrite.   
-
{{:Team:DTU-Denmark/Templates/Footer1}}
+
{{:Team:DTU-Denmark/Templates/EndPage}}

Latest revision as of 12:37, 4 October 2013

Anaerobic production of nitrous oxide from nitrite

Contents

Introduction

In this anaerobic experiment we characterize how readily nitrite is converted to nitric oxide (NO), and subsequently to nitrous oxide (N2O) by adding nitrite (NO2- ) in a series of spikes to:

  • an untransformed strain of E. coli
  • an untransformed strain of P. aeruginosa
  • Mutant 2: a strain of E. coli transformed with the Nir gene from P. aeruginosa
Dtu-Mutant2-extra ws.png

We also monitor nitrite, ammonia and nitrate concentrations before each spike. The concentrations of nitrous oxide is measured continually and the ammonia, nitrite and nitrate concentrations are measured at a series of time points around the spike. For some experiments, we have data for the concentration of nitric oxide as well, however, the sensor frequently gave unreliable results and was replaced during the summer. While we were waiting for the new one to arrive, we were not able to take NO measurements.

Methods

The experimental procedure follows Protocol for Experiment 2.

For the calibration of the N2O and NO electrodes we follow the calibration protocol.

Results and Discussion

In the following diagrams, the y-axis shows mg of Nitrogen (as nitrite, nitrous oxide or other N-source) per litre vs time. Vertical lines indicate the time at which we spiked with nitrite. All experiments were performed anaerobically.

Control: Untransformed E. coli -- Round 1

The first round of the experiment was performed on August 8th and August 9th.

It appears that we observed a response from the cells after the third spike. However, this could also be due to fluctuations in the sensor. DTU-Experiment-2-results.png

Control: Untransformed E. coli -- Round 2

We repeated the experiment to try to reproduce the response that we saw in round 1. The experiment was performed on August 22th. We were not able to see a response in either replicate that we did this round. At the end of the second replicate, the sensor gave a jump in reading, but due to the quick drop off, it is most likely that this is due to fluctuation in the sensor and is not a reliable reading.

Dtu Experiment 2 round 2 --sample A.png Dtu Experiment 2 round 2 -- sample B.png

Untransformed P. aeruginosa

We also did not see a response for the P. aeruginosa, which we would expect.

The experiment was performed on August 7th.

Dtu pseudo exp.png

Nir Transformed E. coli

Finally, we measured both N2O and NO, but did not see an increase in N2O. The NO sensor did not stabilize over the course of the experiment, and these results are not realistic. Additionally, the N2O sensor was calibrated correctly, but when the experimental readings were converted to mg nitrogen, the values were negative. Since this is not realistic, it is likely that the readings are not accurate and that possibly the sensor should be replaced. Unfortunately, we did not have enough time to repeat the experiment with a new sensor.

The experiment was done on the 19th of September and the procedure is described in our notebook for September 19th.

Dtu-nir.png

Conclusions

Despite multiple replicates over multiple experiments, we were not able to reliably detect nitrous oxide being produced in any strain as a result of the addition of nitrite.

Retrieved from "http://2013.igem.org/Team:DTU-Denmark/Experiment2"