The course can be viewed as a series of six related units (see opposite page).
- Boot camp: Students will become acquainted with basic laboratory equipment and techniques for handling and viewing yeast. Students will also be introduced to some of the many online databases, which are important sources of gene and protein information.
- Yeast deletion strains: Students will use selective growth media and the polymerase chain reaction (PCR) to identify three S. cerevisiae mutants, each of which is missing a single gene involved in Met or Cys synthesis (Winzeler et al., 1999). Students will use one of the deletion strains for the remaining experiments of the semester.
- Plasmid identification: Students will isolate yeast overexpression plasmids from bacterial stocks and use restriction endonucleases to identify the plasmids. One plasmid contains theS. cerevisiae MET gene that is missing in their yeast strain (Gelperin et al., 2005). A second plasmid carries the S. pombe homolog for the S. cerevisiae MET gene, and the third plasmid carries an unrelated bacterial gene.
- Transformation and complementation: Students will transform their yeast ∆met strain with the three plasmids. Students will use selective plates to determine if the plasmid-encoded genes complement the met deletions in transformed strains.
- Analysis of protein expression: Students will use western blots to analyze expression of plasmid-encoded genes. The plasmid-encoded proteins are fusion proteins with epitopes at their C-termini that can be recognized by antibodies.
- Team-designed experiment: Teams will design and conduct their own experiments, based on questions that have arisen during the previous experiments.
Overview of the semester’s experiments