Team:DTU-Denmark/Experiments

From 2013.igem.org

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This page summarizes the experiments that we conducted on native and transformed ''E. coli''.   
This page summarizes the experiments that we conducted on native and transformed ''E. coli''.   
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== Initial Characterization of ''E. coli'' ==
Initially, we did two experiments to characterize untransformed ''E. coli'' under conditions that would be present during wastewater treatment.
Initially, we did two experiments to characterize untransformed ''E. coli'' under conditions that would be present during wastewater treatment.
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* Toxicity of Nitrite, Nitrate and Ammonium to ''E. coli''
 
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* Nitrite stability in anaerobic ''E. coli''
 
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In order to verify and characterize our transformants, we performed the following experiments:
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=== Toxicity of Nitrite, Nitrate and Ammonium to ''E. coli'' ===
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* Anaerobic production of nitrous oxide from nitrite
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* Aerobic consumption of ammonium and hydroxylamine
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-
 
+
-
Finally, in order to see if the long term behaviour of our Nir transformant, we grew it in a bioreactor. 
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-
 
+
-
== Toxicity of Nitrite, Nitrate and Ammonium to ''E. coli'' ==
+
Determine whether the ions nitrite, nitrate and ammonium are toxic to ''E. coli'', and at what concentration.  This experiment will use the Biolector to grow an ''E. coli'' strain that constitutively expresses RFP in the presence of these ions (at a range of concentrations) to determine the OD and fluorescence of the cells as a proxy for growth.  We found that at high concentrations ammonium (>=500mM) and nitrate (>=25mM) were toxic to ''E. coli''.  However, these concentrations are much higher than we would expect to find in wastewater, so we do not expect these ions to be toxic to our cells.
Determine whether the ions nitrite, nitrate and ammonium are toxic to ''E. coli'', and at what concentration.  This experiment will use the Biolector to grow an ''E. coli'' strain that constitutively expresses RFP in the presence of these ions (at a range of concentrations) to determine the OD and fluorescence of the cells as a proxy for growth.  We found that at high concentrations ammonium (>=500mM) and nitrate (>=25mM) were toxic to ''E. coli''.  However, these concentrations are much higher than we would expect to find in wastewater, so we do not expect these ions to be toxic to our cells.
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[[Team:DTU-Denmark/ToxicityExperiment|Toxicity Experiment]]
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[[Team:DTU-Denmark/ToxicityExperiment|Experiment details -- Toxicity of Nitrite, Nitrate and Ammonium to ''E. coli'']]
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== Nitrite stability in anaerobic ''E. coli'' ==
+
=== Nitrite stability in anaerobic ''E. coli'' ===
To ensure that any nitrite we produce from Mutant 1 will not be consumed by any of the pathways in native ''E. coli'', we grew ''E. coli'' anaerobically in the presence of varying concentrations of nitrite and measured the amount of ammonia and nitrite present. We found that a pathway to convert nitrite to ammonium is active, and therefore our Nir pathway will need to outcompete this.   
To ensure that any nitrite we produce from Mutant 1 will not be consumed by any of the pathways in native ''E. coli'', we grew ''E. coli'' anaerobically in the presence of varying concentrations of nitrite and measured the amount of ammonia and nitrite present. We found that a pathway to convert nitrite to ammonium is active, and therefore our Nir pathway will need to outcompete this.   
-
[[Team:DTU-Denmark/Experiment1a|Native anaerobic stability of nitrite in ''E. coli'']]
+
[[Team:DTU-Denmark/Experiment1a|Experiment details -- Nitrite stability in anaerobic ''E. coli']]
-
== Anaerobic production of nitrous oxide from nitrite ==
+
== Verification of Transformants ==
 +
 
 +
In order to verify and characterize our transformants, we performed the following experiments:
 +
 
 +
=== Anaerobic production of nitrous oxide from nitrite ===
Grow our transformed Mutant 2 anaerobically in the presence of varying concentrations of nitrite; measure the amount of nitrous oxide produced.  Controls are untransformed ''E. coli'' and ''P.aeroginosa''.   
Grow our transformed Mutant 2 anaerobically in the presence of varying concentrations of nitrite; measure the amount of nitrous oxide produced.  Controls are untransformed ''E. coli'' and ''P.aeroginosa''.   
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[[Team:DTU-Denmark/Experiment2|Experiment 2]]
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[[Team:DTU-Denmark/Experiment2|Experiment details -- Anaerobic production of nitrous oxide from nitrite]]
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== Aerobic consumption of ammonium and hydroxylamine ==
+
 
 +
=== Aerobic consumption of ammonium and hydroxylamine ===
Grow AMO and HAO transformants aerobically in the presence of ammonia; measure concentration of ammonia.
Grow AMO and HAO transformants aerobically in the presence of ammonia; measure concentration of ammonia.
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[[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment 4]]
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[[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment details -- Aerobic consumption of ammonium and hydroxylamine]]
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== Growth of Nir transformant in Bioreactor ==
 
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[[Team:DTU-Denmark/Bioreactor|Bioreactor]]
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== Bioreactor ==
 +
 
 +
Finally, in order to see if the long term behaviour of our Nir transformant, we grew it in a bioreactor. 
 +
 
 +
[[Team:DTU-Denmark/Bioreactor|Experiment details -- Growth of Nir transformant in Bioreactor ]]
{{:Team:DTU-Denmark/Templates/EndPage}}
{{:Team:DTU-Denmark/Templates/EndPage}}

Revision as of 12:02, 29 September 2013

Experiments

This page summarizes the experiments that we conducted on native and transformed E. coli.

Contents

Initial Characterization of E. coli

Initially, we did two experiments to characterize untransformed E. coli under conditions that would be present during wastewater treatment.

Toxicity of Nitrite, Nitrate and Ammonium to E. coli

Determine whether the ions nitrite, nitrate and ammonium are toxic to E. coli, and at what concentration. This experiment will use the Biolector to grow an E. coli strain that constitutively expresses RFP in the presence of these ions (at a range of concentrations) to determine the OD and fluorescence of the cells as a proxy for growth. We found that at high concentrations ammonium (>=500mM) and nitrate (>=25mM) were toxic to E. coli. However, these concentrations are much higher than we would expect to find in wastewater, so we do not expect these ions to be toxic to our cells.

Experiment details -- Toxicity of Nitrite, Nitrate and Ammonium to E. coli

Nitrite stability in anaerobic E. coli

To ensure that any nitrite we produce from Mutant 1 will not be consumed by any of the pathways in native E. coli, we grew E. coli anaerobically in the presence of varying concentrations of nitrite and measured the amount of ammonia and nitrite present. We found that a pathway to convert nitrite to ammonium is active, and therefore our Nir pathway will need to outcompete this.

Experiment details -- Nitrite stability in anaerobic E. coli'

Verification of Transformants

In order to verify and characterize our transformants, we performed the following experiments:

Anaerobic production of nitrous oxide from nitrite

Grow our transformed Mutant 2 anaerobically in the presence of varying concentrations of nitrite; measure the amount of nitrous oxide produced. Controls are untransformed E. coli and P.aeroginosa.

Experiment details -- Anaerobic production of nitrous oxide from nitrite


Aerobic consumption of ammonium and hydroxylamine

Grow AMO and HAO transformants aerobically in the presence of ammonia; measure concentration of ammonia.

Experiment details -- Aerobic consumption of ammonium and hydroxylamine


Bioreactor

Finally, in order to see if the long term behaviour of our Nir transformant, we grew it in a bioreactor.

Experiment details -- Growth of Nir transformant in Bioreactor

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