Team:DTU-Denmark/Experiment4

From 2013.igem.org

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Revision as of 14:28, 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
AMO1 -479.3
AMO2 -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 (TODO add ref) and that even at high throughput, hydroxylamine should not accumulate in the cell.