In this experiment, we will change the genotype and therefore the phenotype of a bacterium by genetically transforming an organism by introducing foreign DNA into its genome. This is called transformation.
In this exercise, we will use the bacterial plasmid as a source of transforming DNA. The plasmid we are using is called pUC 118, and it contains a gene that is resistance to the antibiotic ampicillin. Bacteria that contain the plasmid are able to remain alive and grow in the presence of this antibiotic ampicillin; they are called ampicillin resistant bacteria. Bacteria that do not have the plasmid cannot divide and grow in the presence of ampicillin. You will genetically transform ampicillin-sensitive bacteria (without plasmid) into ampicillin-resistant bacteria (with plasmid). The addition of an ampicillin resistance plasmid will change the genotype of the bacterial cell and will therefore change its phenotype. The transformed cells will gain the ability to synthesize an enzyme that destroys ampicillin.
The E. coli bacteria used do not spontaneously take up DNA. Therefore, the E. coli cells need to be pretreated to make them take up DNA more sufficiently. They need to be "competent" cells. Each lab group will mix DNA with the competent E. coli cells. Any cells that takes up the plasmid DNA and maintains it to express the ampicillin-resistance is called a transformant. One half of cells will be plated on ampicillin plates to determine the number of transformants. The other half of cells will be plated on nutrient agar plates as controls. Each student will count the number of transformants obtained.
1. What is genetic transformation?
2. What is a plasmid?
3. What are competent cells?
4. What are the materials needed for genetic transformation experiment?
5. What are the steps in a genetic transformation experiment?
6. When E. coli cells containing the pUC 118 plasmid are placed on growth medium containing 10 times as much ampicillin as we used in our lab, they are unable to grow.
7. Explain how a change in genotype results in a change in phenotype?