Identifying Functions of Novel Transcripts in S. cerevisiae

Overview

Objectives/Goals Identify the functions of five novel transcripts by replacing with a selectable marker gene (Geneticin resistance). Compare cell growth rate of those with to those without the novel transcript in their genome. Methods/Materials 1)Select the transcripts to study; Microarray charts used to locate position of transcripts on DNA strand. 2)Knock out transcripts with Geneticin resistance gene: a.Amplify the Geneticin resistance gene with primers specific for each transcript, so Geneticin resistance gene would line up and recombine in correct place. b.Transform Geneticin gene into yeast cells. Standard lab transformation protocol followed. Encountered several challenges while working, and had to try five different methods to get it to work. c.Transformation checked by exposure to Geneticin and gel electrophoresis. 3)Analyze and compare the growth phenotypes of the wild type cells and the transformants: a.Tetrad dissection: Break up tetrad (four sister spores) and start a new colony resulting from each spore. b.The deletion found in two of the four spores; yeast cells are diploid, and knockout takes place in only one chromosome. Distinguish between deletion and wildtype by repica-plate exposure to Geneticin. c.Subject both wild type and transformants to different growth conditions and analyze their growth rates. Results We found several differences in growth phenotypes by comparing cell doubling times. However, it is difficult to say if these phenotypes are a result of our knockout or an artifact of the experiment. Conclusions/Discussion The biggest challenge of this project was getting the transformation to work. We spent countless hours troubleshooting and revising our procedure. We realized that this process of constantly revising our methods and looking at the problem from different angles is what science is all about. It made our project interesting, not just a project from a textbook. This realization is what made this project so meaningful to us. Because of time spent on troubleshooting, we did not get to test all the growth conditions we had hoped to. To continue experimenting, we will test the wild type and deletion cells in these other growth conditions in addition to further analyzing the growth phenotype differences we found.

Awards (1)

• Fourth Place

Competition history

• CSEF 2008 Biochemistry/ Molecular Biology · Entry S0409

Resources

• CSEF project summary ↗

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