Throughout our project, we faced many different unexpected obstacles which slowed the progress of our experiment and forced us to adapt. Initially, the plan was to conduct a laboratory-based experiment in which we would measure the masses and concentrations of wild-type and mutant ErbB4 fragments produced through proteolysis by TACE. This experiment would include procedures such as SDS-PAGE (a form of gel electrophoresis) and Western Blotting. We were already slightly behind in conducting our experiment, for multiple reasons. First, we were unable to use the research lab as early as we would've liked because one of the student researchers tested positive for COVID-19, leaving the lab shut down for two weeks. Second, the materials we had ordered for our experiment arrived late due to the winter holidays, which set us back another week and a half. And by the time we were able to begin experimentation, we realized our methodology would be nearly impossible to complete within the given timeframe and deadlines leading up to the Denver Metro Regional Science and Engineering Fair.
The first part of our experimental methods required that we check the cell line (HEK-293) that we were using for the presence of ErbB4 by conducting an SDS-PAGE and corresponding Western Blot without any added enzymes. However, our results were inconclusive and would've required another round of experimentation before we could be certain that the cell line contained ErbB4. In an attempt to speed the process along, our mentors suggested we use a different cell line, MCF-7, which was known to contain the ErbB4 protein. Once again, we conducted the previous experiment with the new cell line as a control, but received even more inconclusive results. Neither round of experimentation was usable in our project. We were right back where we started and running out of time.
While waiting for our laboratory materials to arrive, Kevin and I had begun working on some computational work which could accompany our experimental results in the final presentation. But once our experimental methods were no longer an option, we made the executive decision to shift our project to an entirely computational and theoretical approach. We felt that this was the only way we would finish our project in time for the science fair, and in the end, I believe it was the best decision we could have made. The computational methods would still answer our original question, simply in a theoretical manner using techniques such as sequence alignment and protein visualization in the PyMOL software. Utilizing multiple different computational methods, we not only were able to answer our research question, but we also discovered new information regarding the location of two possible TACE cleavage sites on ErbB4. This information could prove very useful to future experiments on ErbB4 and its ALS-associated mutations, and we never would have been able to discover such information with the purely experimental project we had originally planned. Despite the setbacks we encountered throughout our experimentation, Kevin and I successfully adapted to the situation - just as any real scientist would.
Furthermore, if we choose to continue our project in the future, we could conduct our initial experimental methodology to confirm our theoretical conclusions.