Team:DTU-Denmark/Experiment4

From 2013.igem.org

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== Introduction ==
== Introduction ==
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[[File:dtu-Mutant1.png|right]]
In this aerobic experiment, we add ammonium NH<sub>4</sub><sup>+</sup> to AMO transformed ''E. coli'' cells and measure the consumption of ammonium to determine whether our AMO transformant is converting ammonium to hydroxylamine (NH<sub>2</sub>OH) faster than an untransformed ''E. coli'' control.  A similar protocol is used to determine whether our HAO transformant is converting hydroxylamine to nitrite NO<sub>2</sub><sup>-</sup>, by measuring production of nitrite.
In this aerobic experiment, we add ammonium NH<sub>4</sub><sup>+</sup> to AMO transformed ''E. coli'' cells and measure the consumption of ammonium to determine whether our AMO transformant is converting ammonium to hydroxylamine (NH<sub>2</sub>OH) faster than an untransformed ''E. coli'' control.  A similar protocol is used to determine whether our HAO transformant is converting hydroxylamine to nitrite NO<sub>2</sub><sup>-</sup>, by measuring production of nitrite.
== Methods ==
== Methods ==
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[[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experimental methods]]
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The experiment is based on the protocol [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment 4]] and it was performed on [[Team:DTU-Denmark/Notebook/27_August_2013#lab_115| August 27<sup>th</sup>]] and [[Team:DTU-Denmark/Notebook/28_August_2013#lab_115|August 28<sup>th</sup>]].
== Results ==
== Results ==
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=== AMO Transformant ===
=== AMO Transformant ===
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[[File:Dtu-amo-ammonia.png|600px]]
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[[File:Dtu-rate-amo.png|600px]]
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[[File:Dtu-amo-od.png|600px]]
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Ammonia measured plotted vs optical density from minutes 129 to 885 for two replicates of the AMO transformant, and an untransformed ''E. coli'' control.  The slope (change in concentration of Ammonia / change in optical density) of this graph indicates consumption of Ammonia.
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[[File:Dtu-rate-amo.png|600px]]
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{| class="wikitable"
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! Strain
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! d[NH<sub>4</sub><sup>+</sup>]/dOD
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|-
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|AMO replicate 1
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| -479.3
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|-
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|AMO replicate 2
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| -356.2
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|-
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|''E. coli''
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| -103.2
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|-
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|}
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Ammonia measured plotted vs optical density from minutes 129 to 885 for two replicates of the AMO transformant, and an untransformed ''E. coli'' control.
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Supplemental figures follow.
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[[File:Dtu-amo-ammonia.png|600px]]
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[[File:Dtu-amo-od.png|600px]]
=== HAO Transformant ===
=== HAO Transformant ===
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[[File:Dtu-hao-od.png|600px]]
[[File:Dtu-hao-od.png|600px]]
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Over the course of the experiment, the OD for all treatments that hydroxylamine was added to decreased.  Additionally, no nitrite was detected in any of the treatments.
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Over the course of the experiment, the OD for all treatments that hydroxylamine was added to decreased or remained essentially constant.  Additionally, no nitrite was detected in any of the treatments.
== Conclusion and Discussion ==
== Conclusion and Discussion ==
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The AMO transformant uses ammonia at a rate greater than the untransformed ''E. coli'' control.  This is due to the AMO consuming ammonia and converting it to hydroxylamine.  Additionally, however, the AMO transformant grows significantly more slowly than the control.  
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The AMO transformant uses ammonia at a rate greater than the untransformed ''E. coli'' control.  This suggests that the AMO is consuming ammonia and converting it to hydroxylamine.  Additionally, however, the AMO transformant grows significantly more slowly than the control.  
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It is clear that hydroxylamine is toxic to the cells at this concentration.  With more time, it would be interesting to repeat the [[Team:DTU-Denmark/ToxicityExperiment|toxicity experiment]] using hydroxylamine to determine the threshold concentration that causes cell death.  We expect that ''in vivo'', the hydroxylamine to nitrite reaction catalyzed by HAO  
It is clear that hydroxylamine is toxic to the cells at this concentration.  With more time, it would be interesting to repeat the [[Team:DTU-Denmark/ToxicityExperiment|toxicity experiment]] using hydroxylamine to determine the threshold concentration that causes cell death.  We expect that ''in vivo'', the hydroxylamine to nitrite reaction catalyzed by HAO  
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happens very quickly (TODO add ref) and that even at high throughput, hydroxylamine should not accumulate in the cell.   
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happens very quickly and that even at high throughput, hydroxylamine should not accumulate in the cell.   
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{{:Team:DTU-Denmark/Templates/EndPage}}
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Latest revision as of 19:50, 4 October 2013

Aerobic consumption of ammonium and hydroxylamine

Contents

Introduction

Dtu-Mutant1.png

In this aerobic experiment, we add ammonium NH4+ to AMO transformed E. coli cells and measure the consumption of ammonium to determine whether our AMO transformant is converting ammonium to hydroxylamine (NH2OH) faster than an untransformed E. coli control. A similar protocol is used to determine whether our HAO transformant is converting hydroxylamine to nitrite NO2-, by measuring production of nitrite.

Methods

The experiment is based on the protocol Experiment 4 and it was performed on August 27th and August 28th.

Results

AMO Transformant

Dtu-rate-amo.png

Ammonia measured plotted vs optical density from minutes 129 to 885 for two replicates of the AMO transformant, and an untransformed E. coli control. The slope (change in concentration of Ammonia / change in optical density) of this graph indicates consumption of Ammonia.

Strain d[NH4+]/dOD
AMO replicate 1 -479.3
AMO replicate 2 -356.2
E. coli -103.2

Supplemental figures follow.

Dtu-amo-ammonia.png

Dtu-amo-od.png

HAO Transformant

Dtu-hao-nitrite.png

Dtu-hao-od.png

Over the course of the experiment, the OD for all treatments that hydroxylamine was added to decreased or remained essentially constant. Additionally, no nitrite was detected in any of the treatments.

Conclusion and Discussion

The AMO transformant uses ammonia at a rate greater than the untransformed E. coli control. This suggests that the AMO is consuming ammonia and converting it to hydroxylamine. Additionally, however, the AMO transformant grows significantly more slowly than the control.

It is clear that hydroxylamine is toxic to the cells at this concentration. With more time, it would be interesting to repeat the toxicity experiment using hydroxylamine to determine the threshold concentration that causes cell death. We expect that in vivo, the hydroxylamine to nitrite reaction catalyzed by HAO happens very quickly and that even at high throughput, hydroxylamine should not accumulate in the cell.