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Revision as of 02:11, 18 October 2018
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 next step after protein expression is to characterize the protein activity. The way this was done is by: .
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