Isolating and Characterizing SNARE ts Mutants





Project Summary

At the Bi Lab, a cell biology lab at the University of Pennsylvania, I gained first-hand experience in basic scientific research. Working with a post-doctorate researcher, Hiroki Okada, I examined budding yeast genes that regulate cytokinesis. This related directly to the genetics and molecular biology class I had taken and served as a real-world application for the concepts I had studied. Not only did I develop fundamental lab techniques, but I also acquired skills that extend beyond the realm of research.

The project’s overarching objective is to investigate Inn1, a highly conserved protein thought to be involved in vesicle fusion during cytokinesis. This includes analyzing the SNARE proteins, which bind to Inn1, and comparing mutants of these two proteins. My portion primarily focused on constructing and characterizing temperature sensitive (ts) mutants in the SNARE proteins.

To introduce the mutation into wildtype yeast strains, I performed PCR mutagenesis and transformation. With a lab stock of wildtype SNARE genes and primers carrying the ts mutation, I created SNARE genes that exhibit temperature sensitivity. I then transformed wildtype yeast cells to incorporate the mutated gene. However, the four SNARE genes consist of two pairs of complementary genes: sso1 and sso2, and snc1 and snc2. Each pair is functionally redundant, so to truly see the ts phenotype, I needed to delete the complementary gene. With another PCR mutagenesis and transformation, I replaced the complementary gene with a drug resistant marker. Due to unforeseen obstacles such as low transformation efficiency, I was only able to successfully create mutant strains for sso1 and snc1. This highlighted the inherent uncertainty in research and the constant cycle of trial and error.

After constructing the mutants, I monitored their growth at 25ºC and 37ºC both macroscopically and microscopically. While the cells grow normally at 25ºC, they exhibited phenotypic differences at the higher temperature. There were cell chains and clusters as well as enlarged bud neck regions between mother and daughter cells, which suggests defects in cytokinesis.

However, my greatest challenge came when Hiroki, the post-doc, left for a three-week vacation. He gave me instructions for how to complete the project, but he was not immediately available to answer all my questions. While I had a general outline of the experiments, I needed to work and think more independently. I relied on the other lab members for their scientific knowledge, but ultimately I was the one most familiar with the project. I learned how to interpret unexpected results and overcome them to achieve the research goals. This experience gave me critical thinking and problem-solving skills that have relevance beyond lab work. In addition to gaining scientific knowledge in yeast genetics, I developed greater analytical abilities as well.