Plasmids are small, circular pieces of DNA that replicate independently of the host chromosome. Plasmids have
revolutionized molecular biology by allowing investigators to obtain many copies of custom DNA molecules.
In this lab, you will isolate plasmids from non-pathogenic strains of Escherichia coli, which you will use
in subsequent experiments to transform Saccharomyces cerevisiae met strains.
At the end of this laboratory, students should be able to:
- describe the structure of plasmids and their mechanism of replication.
- identify functional elements that have been engineered into laboratory plasmids.
- explain how the physical properties of plasmids are used in their purification.
- isolate plasmids from transformed strains of Escherichia coli.
Plasmids are the workhorses of molecular biology. Plasmids are small, circular DNA molecules that replicate independently of the chromosomes in the microorganisms that harbor them. Plasmids are often referred to as vectors, because they can be used to transfer foreign DNA into a cell. The plasmids used in molecular biology have been constructed by researchers, who used recombinant DNA technology to incorporate many different functional elements into naturally-occurring plasmids. Plasmids have been engineered to carry up to 10 kb of foreign DNA and they are easily isolated from microorganisms for manipulation in the lab. For the next few labs, your team will be working with yeast overexpression plasmids. Your team will work with three plasmids: a plasmid carrying an S. cerevisiae MET gene, a plasmid carrying itsS. pombe homolog, and a plasmid carrying the bacterial lacZ gene, which will act as a negative control. In this lab, you will isolate plasmids from bacterial cultures. In the next few weeks, you will characterize the plasmids and then use them to transform mutant yeast strains, testing whether MET gene function has been conserved between S. cerevisiae and S. pombe.