Part:BBa_K2272020

pBAD-HAO-Cyt

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Results and Discussion

The pBAD HAO/Cyt BioBrick is designed to oxidize NH2OH to NO2- with the hydroxylamine oxidoreductase enzyme coded by the HAO gene complex. Then the electrons are channeled for this oxidation via the Cytochrome gene complex, namely c554 and cm552, that codes for the Cytochrome enzyme. These coding sequences are extracted from the genome of Nitrosomonas europaea (NE), and for the purpose of our project, are expressed in Paracoccus denitrificans (PD), which is a denitrifier but has demonstrated limited nitrifying capacity.

An experiment was conducted to test the effectiveness of nitrification by PD when transformed with this BioBrick and induced via arabinose. As shown in Figure 1 and based on calculation of the slope values, the transformed PD with the pBAD-HAO-Cyt construct device oxidized ammonium 15% quicker than untransformed PD, but more slowly compared to that of untransformed NE, which naturally oxidizes ammonium. Since our device did not contain the coding sequence for AMO that was responsible for oxidizing ammonium, the transformed PD only relied on its limited natural nitrifying capacity to initiate the nitrification pathway by oxidizing an insignificant amount of supplied ammonium. However, the pBAD-HAO-Cyt was strongly induced by arabinose supplementation and expressed the Hydroxylamine Oxidoreductase enzyme with the Cytochrome enzyme that converted the oxidized ammonium(hydroxylamine) to nitrate persistently. As shown in Figure 2, the transformed PD with the device induced had a drastic increase in nitrate production compared to natural PD or NE, proving that our device functioned properly while being induced. Moreover, the induced device transformed in PD performed the nitrification process more efficiently and quickly than NE did, suggesting that the arabinose-inducible circuit created was not only competent in PD, but also ideal when transformed into PD.

In conclusion, our final device could potentially replace the co-culture of bacteria currently employed in most U.S. wastewater treatment plant sewage sludges, and even work better than the current methods.

Figure 1. This graph compares the efficacy of performing nitrification of our model organisms, Paracoccus Denitrificans and Nitrosomonas Europaea, versus our transformed chassis organism, Paracoccus Denitrificans with the HAO-CYT circuit. Efficacy was measured in change in the concentrations of ammonia over time, which showed efficiency of Nitrification in our Bioreactor.

Figure 2. This graph compares the efficacy of producing nitrates, an end product of the nitrification process, between our model organisms, Paracoccus Denitrificans and Nitrosomonas Europaea, versus our transformed chassis organism, Paracoccus Denitrificans with the HAO-CYT circuit.

Experimental

Cells were pre-cultured and cultured in ammonium rich media before being spiked with an ammonium chloride solution. The final culture reached a 50mM [NH4], on par with many wastewater facility influent conditions. [NH4], [NO3], and [DO] concentrations were then measured at two minute intervals along with OD600 readings at thirty minute intervals.

1L of ammonium-rich culture reached OD 0.3. Culture split into three 294ml aliquots. Three volumetric flasks labeled Flask 1, 2, and 3 each receive one 294ml aliquot. Initial ODs of each 294ml flask taken. Initial [NH4+ ], [NO3+], and [DO] recorded for each flask at two minute intervals. At t=0 minutes, 6ml [NH4] spike solution added to each flask. Theoretical [NH4] of each flask brought to 50mM, an established value for average [NH4] in wastewater treatment plants. [NH4], [NO3], and [DO] measured in each flask at intervals of two minutes. At each two minute, three measurements are taken, one for each flask. After the measurement, each sensor is washed with deionized water and rotated to the next flask. This experimental design staggers measurements so that accurate changes in nitrogen containing compounds are measured. Rotation and measurement proceeds until t=90.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

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• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1572
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  Illegal NgoMIV site found at 3821
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  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1476
  Illegal BsaI.rc site found at 1524
  Illegal BsaI.rc site found at 2091
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  Illegal BsaI.rc site found at 5928
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