Techniques for transforming microbial organisms with foreign DNA are essential in modern molecular biology. In this lab,
you will transform a S. cerevisiae met strain with three different plasmids and use ura3complementation to detect transformed
cells. You will then use replica plating to determine if S. pombe Met genes are functionally equivalent toS. cerevisiae MET genes.
At the end of this lab, students should be able to:
explain the processes of transformation and complementation at the molecular level.
design a selection strategy to isolate transformed strains
transform S. cerevisiae with plasmids and isolate transformants on selective media
use replica plating to analyze the ability of plasmid-encoded MET genes to complement met deficiencies
In this experiment, you may receive a preliminary answer to the semester’s research question about the functional conservation of Met proteins in the Ascomycota . During the first part of the semester, your team used selective plating and colony PCR to identify yeast deletion mutants. You then isolated and characterized plasmids that can be used to overexpress Met proteins. These two sets of experiments come together in this lab, when you transform the S. cerevisiae deletion strain with the expression plasmids. Through a series of complementation experiments, you will determine if the genes carried on the plasmids are able to compensate for the missing MET genes in the mutants. In complementation, the introduced gene restores the normal phenotype to a mutant with a defective gene.