Experiment 2: Analysis of phosphate acquisition mutants of E. coli . Present the

Question

Experiment 2: Analysis of phosphate acquisition mutants of E. coli.

Present the table from the handout on which you recorded your results. (No marks, will be used as a guide for marking Q2)

Strain

Growth on Pi plates(yes/no)

Growth on G3P plates (yes/no)

AP-ase assay colour

1

No

Yes

Yellow

2

No

Yes

None

3

Yes

Yes

Yellow

4

Yes

Yes

None

Use your results to determine the genotype of each strain (bacteria are haploid, so you would use the terms “(loss of function) mutant” or “wild type”) and complete the table below. (1 mark)

Genotype

Strain

Alkaline phosphatase

Pi transporter

1

2

3

4

Explain how you reached your conclusions for the genotype of each strain. (1 mark)

Why is growth on G3P is an important control for this experiment? (2 marks)

Suppose the DNA sequences for the alkaline phosphatase and the Pi transporter genes from strain 1 were determined. If both genes are found to contain a single base change compared to the wild type sequences, what could you conclude about the effect of each mutation on the encoded protein? (2 marks)

Strain

Growth on Pi plates(yes/no)

Growth on G3P plates (yes/no)

AP-ase assay colour

1

No

Yes

Yellow

2

No

Yes

None

3

Yes

Yes

Yellow

4

Yes

Yes

None

Explanation / Answer

Genotype

Strain

Alkaline phosphatase

Pi transporter

1

wild type

mutant

2

mutant

mutant

3

wild type

wild type

4

mutant

wild type

1.Explain how you reached your conclusions for the genotype of each strain.

Ans: Where mutation occurred transporter or alkaline phosphatase became inactive. Thus, in first two strains 1& 2 unable to grow in Pi containing plate and two strain 2& 4 unable to generate colour in AP-ase assay.

2.   Why growth on G3P is is an important control for this experiment?

Ans: In presence of G3P or glyceraldehyde-3-phosphate all strains showed growth. As, G3P used in in glycolysis of bacteria that process occurs in all the plates thus in all cases bacterial strains are alive, their other biological activities are normal, only they have mutation in Pi transporter or alkaline phosphatase gene in specific strains.

3. Suppose the DNA sequences for the alkaline phosphatase and the Pi transporter genes from strain 1 were determined. If both genes are found to contain a single base change compared to the wild type sequences, what could you conclude about the effect of each mutation on the encoded protein?

Ans: If in one base has been changed for both the protein in strain 1 then it is a silent mutation (mutation does no effect on coding amino acid sequence) occurred in case of alkaline phosphatase where as missense ((mutation changes amino acid sequence) or nonsense (mutation creates a stop codon) mutation occurred in case of Pi transporter.

Genotype

Strain

Alkaline phosphatase

Pi transporter

1

wild type

mutant

2

mutant

mutant

3

wild type

wild type

4

mutant

wild type

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