Team:WLC-Milwaukee/Demonstrate
Results
Cloning
The wet lab team began cloning our parts in early summer, and according to the data, achieved successful clones.
An agarose gel, ran on July 8, 2019. In wells 2 and 3 was a sample of DNA after ligation containing the pbrR promoter and BSMT 1. In lane 4 was a sample of DNA after ligation containing the pbrR, the T7 promoter, and BSMT1. The other lanes were filled with the same DNA samples.
From this gel, we achieved successful clones of pbrR and BSMT1 on one plasmid and more successful clones of pbrR, T7, and BSMT1. However, we did not get confirmation of any successful clones of pbrT. Therefore, we continued with cloning to attempt to get every gene into the same plasmid.
A DNA agarose gel ran on August 6, 2019. In lanes 18 and 19 are DNA samples of the pbrT ligated to the plasmid psb1c3. In lanes 9, 10, and 11 are DNA samples containing pbrT, pbrR, BSMT1 genes, as well as the T7 promoter.
In this gel, we obtained confirmation that we had successfully cloned pbrT, pbrR, BSMT1, and the T7 genes into the same plasmid. We transformed this plasmid into the E. coli and verified our results by sending cultures of the transformed E. Coli for sequencing. The sequencing results confirmed that we had successfully ligated and transformed our predetermined genes into our E. coli chassis organism.
Next, we attempted to verify that the pbrR, pbrT, and BSMT-1 proteins were being produced by performing a western blot. At first, our western blots were unsuccessful due to old secondary antibodies. However, after purchasing new antibodies, we began to see interesting data.
A western blot performed on the morning of October 5, 2019. Flag tag antibodies were put on the top blot, and histidine antibodies on the middle blot. The bottom blot had antibodies from the tolC gene. We loaded four wells with our successfully transformed E. coli along with varying concentrations of lead. In the wells, reading left to right were 0ppb, 25ppb, 50ppb, and 100ppb of lead along with our transformed E. coli.
This western blot was unsuccessful in determining if the correct proteins were being produced or not. While there are proteins showing up on the blot, they were in the incorrect places. This means that the proteins showing up were larger or smaller than what we expected. We attempted another western blot.
A western completed in the morning of October 11, 2019. Histidine antibodies were added to the top blot, tolC antibodies to the middle blot, and flag-tag antibodies to the bottom blot. 0ppb, 25ppb, 50ppb, and 100ppb lead samples, along with our transformed E. coli was added to wells 2-5, respectively. Additionally, 100ppb lead with E. coli competent cells was added to the sixth well.
This western blot also failed. Again, bands showed on the developed film, but in the wrong places. This led us to conclude that, despite the successful transformation of the genes responsible for protein synthesis. Our test kit is not producing the correct proteins to produce the wintergreen scent and is therefore an ineffective method in determining the amount of lead present in a given sample of water.
