Genetics Experiment

Experiment 3: Characterization of Nonsense Mutations in E.Coli

A. Purpose:

a. This experiment was conducted with lac- mutants mated with F- strains with a lac-pro deletion region with different amber suppressors. These matings were then tested to determine if the amber suppressors of the F-strains suppress the nonsense mutations on the lac- mutants. Each amber suppressor gene recognizes the stop codon UAG and inserts a amino acid to restore function of the protein.

B. Controls:

a. The controls are lac- strain on min +strep and min+nal plates, CSH54 on min+strep plate, CSH55 on min+nal plate, and CSH56 on min+nal plate. The controls are used to test if the strains do indeed contain the correct properties.

b. The lac- strain has proline but is sensitive to nalixidic acid and streptomycin. Hence, there should no growth on the control plates for lac- mutants. The CSH54 strain is resistant to streptomycin but lacks proline and lactose. So though it may be able to survive in the antibiotic environment on the min+strep plate, it should not grow due to its lack of proline. The CSH55 and CSH56 strains also lack the proline-lactose region but both are resistant to nalidixic acid. Thus, there should be no growth on the min+nal plate because both strains lack the proline gene.

c. The lac- mutant and the CSH54 control plate did not exhibit any growth. The CSH55 and CSH56 strains exhibited 15 and 7 colonies, respectively. This growth could be due to the fact that the CSH55 and CSH56 strains have a semi-functional lac gene.

C. Results:

Table 1. Matings on Min + Lactose plate

Mating Observations

F (lac- with CSH54) No growth

E (lac- with CSH55) No growth

D (lac- with CSH56) No growth

Lac+ control White colony

Lac- control No growth

Table 2. Matings on MacConkey’s Agar.

Mating Observations

F (lac- with CSH54) 8 white colonies

E (lac- with CSH55) 6 white colonies

D (lac- with CSH56) 6 white colonies

Lac+ control 1 purple colony

Lac- control 1 white...