Experiment 3
-Evaniya Shakya
Purpose
Hfr lac- E. coli were mated with 3 different F- strains that had deletion of lac and pro genes. These F- strains also contained nonsense suppressor genes for the UAG codon. The purpose of this experiment is to determine if the Hfr lac- strain has a nonsense mutation and if this mutation can be suppressed by any of the three suppressor tRNAs: supD, supE and supF. We wanted to see if the substituted amino acid would restore the function of the protein.
Controls
We plated:
• Hfr lac- on min+ strep and min+ nal- the Hfr lac- is pro + but doesn’t have resistance against streptomycin and nalidixic acid so no growth is expected.
• CSH54 on min+ strep – This strain is pro- and streptomycin resistant. However, there should be …show more content…
• Exconjugants from mating D grew on min + lac plate and were red on Maconkeys showing that it has the ability to use lactose. So it can be concluded that Hfr lac- had a nonsense mutation(UAG). Through the mating, the exconjugant received the gene to make supD tRNA that was successful in suppressing the STOP codon UAG with the amino acid serine. The substitution of serine in the amino acid chain does not harm the protein function or structure. Therefore, the bacteria is successful in utilizing lactose properly.
• For exconjugants from mating E there was no growth on min+lac and Mackonkeys had white colonies. So supE which used glutamine to suppress the UAG nonsense mutation was not successful in creating a protein that can utilize lactose. The substitution of glutamine may have severely changed the structure/function of the protein so it could still not use lactose. So, there was no restoration of function.
• Exconjugants from mating F showed some faint growth on min +lac and White with