Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes, Blog Post #2

We are still in the initial stages of our research, which means we have not yet perfected our systems and procedures. Arianna, Professor Chang and I spent time working on a poster which was presented at the 2018 Gordon Research Conference on Biomineralization, held at Colby-Sawyer College in New London, New Hampshire from July 28 – August 3. My section of the poster required me to search for possible uses of biosilica prepared with silicatein. To do so, I read through literature found during our search using Web of Knowledge. I learned about the possible uses of biosilica in fields such as regenerative medicine, as well as 3D printing. I also came to the conclusion that there is not much information out there on our specific studies which limited the contribution of future uses I could add to the poster.

As far as lab work, we have worked on creating a successful method in which we can express protein. Along the way we ran into some issues. Our group started with trying to express a protein called lactate dehydrogenase from the Mackerel ice fish (c. gunnari). Our first system included a pUC57 vector purchased from GeneWiz containing the cgLDH gene which we hoped to express in the DH5-ɑ e. coli strain purchased from New England Biolabs (NEB). Unfortunately, we were unsuccessful this attempt, due to the fact that the construct was not compatible to express our cgLDH protein. For our next attempt we used a pET11a vector with the cgLDH gene inserted from GeneScript and T7 Express e. coli from NEB. When collecting data at the end of our expression system, we initially thought we had once again not produced any protein because measurements using a NanoDrop 1000 spectrophotometer implied protein was not present. After Professor Chang performed a more sensitive assay, he concluded we did indeed produce the protein, although in very small quantities. I am curious as to why we are not seeing the amount of protein one might expect, and which step of the system is hindering this production.

I believe in the future we need to modify our procedure in order to optimizing growth conditions to produce the protein in large quantities.The expression of cgLDH is important for a research project that is in collaboration with research groups at Albert Einstein College of Medicine. Thus, even though cgLDH is not directly related to our current project, it is in our hope that cathepsin-L can be expressed using T7 Express e. coli and the pET11a vector as well.

I have certainly learned a few lessons from this project. I have learned that in research there is a lot of trial and error that goes on, and sometimes you can work for hours or even days on a single thing and have it be unsuccessful. I’ve learned from Professor Chang’s example that you can’t let these setbacks throw you off or deter you from trying a new procedure. I’ve also learned that it is important to use your resources and have those who are experts with certain materials help you if you are unsure where the process is failing. Our experience this summer leaves me determined to perfect our protein-producing system in the future so we can continue with our project.

 

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