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

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