Team:Virginia/Results
Results
Characterization of Chassis
Paracoccus denitrificans
There were two attempts to generate a well-characterized growth curve for P. denitrificans under our laboratory conditions. The first resulted in a culture reaching a growth plateau at an OD600 of around 0.4. The growth plateau was reached between five and six hours after inoculation into a 1 L liquid culture in PD media (see protocol/link). Growth was completely plateaued by ten hours. The error bars shown are 95% confidence intervals. The OD600 values of this round of measurement was done with a NanoDrop machine that was potentially not capable of accurately determining the cell density.
The second attempt yielded extremely different results. Again, the culture was grown in a shaking incubator at 33.0 C at 220 rpm. The same PD media was used to grow the culture. The plateau was at an OD600 between 2 and 2.5, Exponential growth ended at 10 hours just as it did in the first trial. Mid-exponential growth was reached at four to five hours just as in the first trial as well. This round of measurement was done with Spectronic Genesys 2, which detected the OD600 of the cell culture more precisely than NanoDrop did.
The second attempt was unsuccessful. The culture reached a plateau at an OD600 of ~0.15 before gradually falling to 0.13. This failure was indicative of the challenge in maintaining a stable P. denitrificans culture. Growth curves for N. europaea were not created as we could not culture it successfully in time to generate a growth curve.
Summary
Our summer project was designed to improve the process of nitrogen removal in the tertiary sewage treatment stage of raw sewage processing. Currently, a fairly common design is to spatially separate the processes of nitrification and denitrification so that distinct bacterial populations within activated sludge can perform their respective biochemical processes independently. Our project proposes that both processes be temporally separated and housed under a single organism. To achieve this, we attempted to transform the genes encoding for proteins responsible for nitrification into Paracoccus denitrificans, a known denitrifier in active sludge.
By the end of summer experimentation, we were able to transform most, but not all of the nitrification genes into Pc. denitrificans. Our central composite circuit consists of genes for HaoA and HaoB, proteins that catalyze the oxidation of hydroxylamine into nitrite ions. It also includes CycA and CycX, genes encoding for two c-type cytochromes involved in shuttling electrons from the ammonia oxidation reactions to terminal electron acceptors. We were, however, unable to prepare the genes for AmoA, B, and C into Pc. denitrificans. These genes would have encoded for a heterotrimeric protein complex that would have catalyzed the oxidation of ammonia into hydroxylamine.
In addition to the ammonia oxidation circuit we developed, we also designed a series of reporter circuits to test the function of several promoters in P. denitrificans compared to E. coli. We were able to transform Pc. denitrificans with a composite part consisting of Mn-SOD, an oxygen-inducible promoter, and GFP. With this reporter circuit, we attempted to characterize promoter activity in response to different dissolved oxygen levels by measuring relative fluorescence at the absorbance spectra of GFP compared to control untransformed cells.
In addition to Mn-SOD, a new promoter added to iGEM, we also tested the promoter activities of VhB and T7 in transformed E. coli cells. These promoters were connected to the coding sequences for GFP in a composite part. Again, all promoters were tested at various dissolved oxygen levels. These were tested against untransformed E. coli cells. Finally, we accounted for the fact that different oxygen levels may lead to different growth rates, so we divided RFU values by the OD600 of a given sample that was measured.
