experiment to find out if promoter differences causing repressor binding issues?

Question

I don't have much research experience, and I need to design an experiment to figure out if promoter differences in different strains of a certain bacterium affect the way a repressor protein is able to bind to the promoter. Any thoughts?

actually, i don't know the repressor protein. I just need to show it may exist and be the reason why one strain produces more of the relevant protein than the other stain.

also, how do you go about isolationg the promoter? Thanks.

Answer 1

I'd clone the promoter from each strain and link it to Green Fluorescence Protein. I'd also clone the repressor protein gene and place in an expression cassette and transform the whole works into bacteria and measure the levels of GFP in each. You can also run binding assays, but if the DNA sequences are known, or at least primers on the 5' and 3' end are known for the above promoters and repressor protein gene, I'd do the cloning. If your lab is more into protein purification then run the binding assays.

Answer 2

An easy experiment is to first obtain the promoter sequences of each strain of bacteria.

Then, compare which bacteria strains has the highest affinity of the repressor protein to the promoter and you can mutate the promoters of other bacterial strains so that they can resemble other strains' promoters.

For example, if you have two strains with two different promoters which bind the same repressor proteins (one binds tightly, the other loosely) then if you take the 'tight' promoter and replace it with the 'loose' promoter you should be able to observe a decrease in binding affinity of the repressor protein to that promoter.

-Kevin

Answer 3

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