Question

Glutamate synthetase is central to nitrate assimilation. You, as a grad student, are curious about how other nutrients might regulate this enzyme; and specifically, you would like to study how the gene encoding this enzyme is regulated by the presences of phosphate in the environment. Describe the design of your experiment(s) that will look at the regulation of the glutamate synthetase gene by phosphate. Remember to include controls in your description.

Answer 1

You can make media containing Glucose as a sole carbon source, potassium nitrate as a nitrate source and variable phosphate sources (potassium phosphate and calcium phosphate) to confirm if there is change in expression of gene is only due to phosphate.

methodology

you can make 3 types of tubes

1 : glucose + potassium nitrate

2 : glucose + pot. nit. + potassium phosphate

3 : Glucose + pot. nit. + calcium phosphate

Tube 1 will be the control of the experiment as no phosphate source is added.

You can do Real time qRT-PCR using specific primers for glutamate synthase mRNA to check the expression level of this gene in each condition. This will give you the amount of mRNA generated in each condition. If the level of mRNA is higher in presence phosphate compared to control you can say that phosphate is inducing the expression of tge gene.

You can confirm it by extraction of glutamate synthase enzyme from each conditions. You can purify enzyme using affinity chromatography. Then you can measure the concentration of enzyme extracted from each conditions and you can run SDS-PAGE to check purity of extracted enzyme.