Bootstrap Example

Cloning Wild Type HRP

Part of the project involved the transformation of PFL-HRP into Saccharomyces Cerevisiae strain BJ5464 with a Gal1 Promoter. The data we gathered suggested we were successful in this endeavor.

PCR Digest

Plasmid Sequencing

After confirming that we have a possible success, we extracted the shuttle vector DNA and submitted it for sequencing. The results of which can be seen below.

HRP characteriztion and expression

Protein expression using a protein gel

After confirming the transformation of the HRP, the next step is to characterize the activity of the HRP. Since the HRP was under the operation of Gal1, in order to induce expression, it needs to be grown on a media with galactose media. Then in order to confirm the functioning of the promoter, it would be compared to yeast grown on a plate without galactose such as glucose or YPD. Lysate from colonies on these media was collected and then lysed. This lysate was ran on a protein gel which can be seen here.

The protein gel suggests that there might be HRP in the lysate, however it is also very apparent that there are many different proteins in the lysates.

Protein characterization

The simplest way to test for the presence of functional HRP is to mix reagents necessary for a colorimetric into the sample of interest; in our case, the sample is full-cell lysate and the reagents include TMB and H2O2. To prepare samples, 50mL cultures of our transformed yeast were inoculated in both galactose and glucose media. When initial tests failed to demonstrate HRP production in the induced sample we investigated potential causes. Using store-bought samples of HRP, we tested the colorimetric reaction at different concentrations of lysate. Using constant final concentrations of HRP, TMB, and H2O2 we discovered that an increase in lysate concentration corresponds to an apparent decrease in the extent of reaction. Presumably, something in the lysate inhibits the reaction from occurring. Our advisors indicated that native yeast proteins like catalase or superoxide dismutase could potentially consume the added H2O2 before HRP has the opportunity to react. With this information we added copper ions, an inhibitor to these proteins, to the cell lysates at a final concentration of 50mM in the hopes of witnessing an HRP-based color change. Although evidence suggests the ions were successful in inhibiting the native proteins, they also reacted with the lysate to produce a precipitate and an unexpected color change..