Experiment 3: Characterization of Nonsense Mutations in E.Coli
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.
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 ...view middle of the document...
Matings on Min + Lactose plate
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.
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 colony
D. Conclusion and Explanation:
a. In order to determine if the mating were initially successful, all the matings were plated on minimal media + antibiotic plates. The antibiotic resistance gene from the F- strains would allow the matings to grow in the antibiotic environment. The lac- mutants would provide pro+ gene so that the matings could grow on minimal media. Thus only successful matings of the strains through conjugation would be able to grow on the min + antibiotic plates. During gene transfer, the lac- strain transfers the lac-pro region early and the suppressor regions (supD, supE, supF) late. Since crossovers can only occur between homologous parts of DNA and the F- strains have a deleted lacpro region, there is a double crossover event on homologous points on either side of the lacpro region. Hence, it is assumed that any successful mating between the Hfr strain and the F- strain will contain both the lac- and pro+ genes. On the MacConkey’s plate, only the lac+ control was pink/purple looking because it was the only strain that metabolized lactose present in the medium. Since all the matings F, E, and D and the lac- control were white, they did not contain the lac+ gene. Since all the lac- exconjugants (F, E, and D) did not grow on the min +lac plate, the mutation was either a missense mutation that was not suppressed by the supD, supE, and supF genes or a nonsense mutation that was not recognized by any of the amber suppressor genes. The lac- control did not grow because it lacked the gene necessary to metabolize lactose. If there was growth for all the matings on the min+lac plate, it means that serine, glutamine or tyrosine was inserted for the stop codon UAG in strains D, E, and F respectively thereby suppressing the nonsense gene. The supD, supE, and supF are suppressor genes that recognize the amber stop codon and restore function of the protein.