E14 - E1. A. -ONPG was used to measure the level of...

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E1. A. ß -ONPG was used to measure the level of expression of the lac operon. More specifically, the cleavage of ß - ONPG to produce a yellow compound requires the action of ß -galactosidase. So the assay is indirectly measuring the amount of ß -galactosidase protein. The reason why there was no yellow color in one of the tubes was because the repressor was preventing the expression of the lac operon, and this prevents the expression of ß -galactosidase enzyme. Some other methods to measure the expression level of the lac operon could include the following: 1. Conduct a Northern blot to measure the amount of mRNA that is produced from the lac operon. 2. Conduct a Western blot using antibodies against one of the three proteins encoded by the lac operon. 3. Measure the uptake of radiolabeled-lactose into the cells. This would measure the amount of the lactose permease that is expressed from the lac operon. B. The merozygote has two copies of the lacZ gene so it makes twice as much ß -galactosidase. E2. In samples loaded in lanes 1 and 4, we expect the repressor to bind to the operator because there is no lactose present. In the sample loaded into lane 4, the CAP protein could still bind cAMP because there is no glucose. However, there really is no difference between lanes 1 and 4, so it does not look like the CAP can activate
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This note was uploaded on 04/06/2009 for the course BIO 325 taught by Professor Saxena during the Spring '08 term at University of Texas.

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E14 - E1. A. -ONPG was used to measure the level of...

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