Experiments
In order to test whether or not our solution would work, we had to conduct some experiments on the effectiveness of the bacteria. We conducted these experiments at Genspace and BUGSS, with each location focusing on different parts of the project.
Genspace
The experiments at Genspace focused on creating a bacteria which would bioluminesce in the presence of PCBs.
Experiments
The goal of this part of the project was to transfer a plasmid containing the Akaluc construct into the chassis of choice, which was yeast. Towards the end of the project, we also focused on increasing the visibility of the bioluminescence, though we did not get very far on that.
Results
Through repeated ligations, purifications, transformations, and platings, we eventually achieved cells which contained the desired gene.
BUGSS
The experiments at BUGSS focused on the other aspect of the project, that being the degradation of PCBs into less harmful products.
Experiments
This portion was cloned a plasmid in E. coli and then transferred it into Rhodococcus jostii as a chassis, since it can survive in water and on land.
Results
We were able to successfully clone pcbA4 into E. coli as shown by the band of ~1500 bp on a colony screening PCR gel.
Due to time constraints we did not test the efficacy of our degradation genes, pcbA1, pcbA4, and pcbA5. If we were to continue this project in the future, we would experiment with the reaction rates of those enzymes on the breakdown of PCBs.
Experiments on Rhodococcus
Because Rhodococcus spp. do not have a lot of documentation in iGEM, we also focused on characterizing the bacteria. In addition to transforming the bacteria to include our desired genes, we also tested for its optimal growth conditions and growth rate. We ran each test multiple times, changing conditions slightly each time, in order to identify the best environment for this bacteria.
Results with Rhodococcus
From our experiments with Rhodococcus jostii, we have learned a lot more about this species of bacteria. First, it seems to prefer a temperature of 30 degrees Celsius, which is slightly colder than E. coli. Finally, it has a doubling time of about 190 minutes. Also see our Contribution page for more characterization of Rhodococcus growth.
