Team:DTU-Denmark/Experiment2

From 2013.igem.org

(Difference between revisions)
(Methods)
(Introduction)
 
(43 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 ==
-
'''Measuring the production of N<sub>2</sub>O from Nitrite (NO<sub>2</sub><sup>-</sup> ) anaerobically'''
+
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''
-
We grew ''E. coli'' and ''P.aeroginosa'' anaerobically in the presence of varying concentrations of nitrite and measured the amount of nitrous oxide produced.
+
* an untransformed strain of ''P. aeruginosa''
-
The behavior of ''E. coli'' (untransformed cells) and ''P. aeruginosa'' is characterized while growing in the presence of NO<sub>2</sub><sup>-</sup> in an anaerobic reaction chamber by measuring the conversion of nitrite (NO<sub>2</sub><sup>-</sup>) to nitric oxide (NO) and nitrous oxide (N<sub>2</sub>O).
+
* Mutant 2: a strain of ''E. coli'' transformed with the Nir gene from ''P. aeruginosa''  
-
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.
+
[[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.
-
== Methods ==
+
==Methods==
-
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<sup>o</sup>C.
+
The experimental procedure follows [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_2|Protocol for Experiment 2]].
-
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).  
+
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]].
-
Additionally, we will measure the response of only anaerobic ''E. coli'' biomass without any NO<sub>2</sub><sup>-</sup> added, and the same for anaerobic ''P. aeruginosa''.  This will be done in the experimental flasks prior to spiking with  NO<sub>2</sub><sup>-</sup>.
+
== Results and Discussion ==
-
There will be 2 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.
-
# ''E. coli'', to which spikes of NO<sub>2</sub><sup>-</sup> will be added.
+
=== Control: Untransformed ''E. coli'' -- Round 1 ===
-
# ''P. aeruginosa'', to which spikes of NO<sub>2</sub><sup>-</sup> 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 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>]].
-
The response time of the cells to spikes of NO<sub>2</sub><sup>-</sup> is expected to be on the order of minutes, and we will run the experiment until the solution has reached saturation with NO<sub>2</sub><sup>-</sup>.
+
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]]
-
'''EQUIPMENT NEEDED'''
+
=== Control: Untransformed ''E. coli'' -- Round 2 ===
-
*1 400 mL bottle with a lid that the electrodes can be fastened air tightly to
+
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 magnetic stirrer
+
-
*1 NO probe for NO measuring
+
-
*1 N<sub>2</sub>O probe for NO<sub>2</sub>O 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
+
-
'''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'' ===
-
# Prepare DM minimal medium with ammonium chloride as a nitrogen source.  Add 0.745 g NH<sub>4</sub>Cl to 1L of prepared DM medium. 
+
-
# Start overnight cultures.
+
-
# Prepare test solutions for ammonium, nitrite and nitrate kits.
+
-
# Label eppendorf tubes and test tubes for colorimetric samples.
+
-
# Grow ''E. coli'' top10 overnight/ ''P. aeruginosa'' in 10mL of DM medium + NH<sub>4</sub>Cl prepared in step 1 at 37<sup>o</sup>C.
+
-
# Take 4mL of ''E. coli''/''P. aeruginosa'' overnight culture and add to 200mL fresh DM medium + NH<sub>4</sub>Cl.
+
-
# Grow the cells at 37<sup>o</sup>C in 200 RPM until OD=0.35 (about 3 hours; about 5 hours for P. aeruginosa).
+
-
# Pellet down the 200mL culture, 3000g for 4 min (Cool down the centrifuge if it is needed for 30 min at 4◦C).
+
-
# Wash with 5 ml cold Modified DM minimal medium and centrifuge again.
+
-
# 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.
+
-
# Measure OD of the 200 mL cell suspension and add Modified DM minimal medium until OD=0.3 (note the exact value).
+
-
# Pure the 200 mL of the OD=0.3 suspension to a bottle.  This is the experimental flask. 
+
-
# For the control: Remove one aliquot of 200mL of DM medium (without the added NH<sub>4</sub>Cl). 
+
-
# Make the anaerobic experiment by saturating with pure N<sub>2</sub> following the method for injecting N<sub>2</sub> ''(Appendix 1)'', for 5 min the nitrite stock solution and 10 min the cell suspensions and controls.
+
-
# Start the experiment by adjusting the NO and N<sub>2</sub>O electrodes in one flask at a time.
+
-
# Put the flask on the magnetic stirrer and start with 270 rpm at 37<sup>o</sup>C.
+
-
# Remove 2 mL as the first sample t=0 colorimetric analysis.
+
-
# Add 0.5 mL of 50mM nitrite stock solution to 100 ml of the cell suspension.
+
-
# Watch the concentrations of NO and N<sub>2</sub>O, and continue adding nitrite when they are not changing (about 10 min).
+
-
# Repeat steps 18-20 until the solution exceeds the sensitivity of the sensor (1mM).
+
-
# Make the colorimetric measurements for nitrite, nitrate and ammonium for each of the samples collected by using the appendices 3 and 4 in order to finish gathering data.
+
-
== Results ==
+
We also did not see a response for the P. aeruginosa, which we would expect.
-
[[File:DTU-Experiment-2-results.png|600px]]
+
The experiment was performed on [[Team:DTU-Denmark/Notebook/7_August_2013#Lab_115|August 7<sup>th</sup>]].
 +
 
 +
[[File:Dtu pseudo exp.png|600px]]
 +
 
 +
=== Nir Transformed ''E. coli'' ===
 +
 
 +
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.
 +
 
 +
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-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.