Team:UrbanTundra Edmonton/Results
Results
The expression of perchlorate dismutase
For the expression of our Pcr construct, we chose to use the iGEM submission vector PSB1C3. Using the Golden Gate ligation method, a single restriction enzyme, BsaI, was used to assemble the gBlocks in the correct order. The BsaI sites, when cut by the restriction enzyme, leave non-complimentary non-palindromic ends. Due to this, the pieces of our construct would anneal in the orientation that we desired: LacIq, PcrA, and PcrA/B.
Transformation of Pcr
The first attempted transformation into DH5α E. coli was unsuccessful. Using a chemical transformation protocol, we were unable to successfully incorporate our plasmid into the competent cells, producing no colony growth on our plates. In discussion with the UAlberta iGEM team, they suggested to try two different things: an electroporation protocol for the transformation of our plasmid into competent cells as well as to use DH10B E. coli as our chassis. In one of our many collaboration efforts with the UAlberta team, they aided us in preforming an electroporation on both DH5 and DH10B E. coli. Fortunately, this transformation yielded colonies for both chassis, albeit only a few.
Future Steps
To validate the effectiveness of our part, a screening test involving perchlorate would be conducted. As we found out in our research from last year, chlorite is more toxic than perchlorate, even at extremely small concentrations. In other words, it is easier for E. coli to survive in perchlorate than in chlorite. The contrast in toxicity can be used to determine whether our construct was successfully incorporated into our plasmid. We would do this by plating the E. coli that contains our plasmid onto plates that contain the threshold amount of perchlorate along with growth media, which is around 0.1 M. If our part works, the bacteria would produce perchlorate reductase and would convert the perchlorate on the plate into chlorite. Due to chlorite’s toxicity, it would kill the chassis, preventing significant growth on the plate. Essentially, a plate that showed dead E. coli would verify that our part worked.
References
UrbanTundra_Edmonton 2016. “Experimental” 2016.igem.org/Team:UrbanTundra_Edmonton. 2016.
