You are analyzing an unknown gene that is expressed robustly

You are analyzing an unknown gene that is expressed robustly under certain conditions. You have sequenced the gene and in the 5\' UTR of the mRNA you find the following sequence: 5\'-CGCCGCCGCCGCCGC-3\'. You cleverly replace the unknown gene with a gene that you can monitor in several ways (this is called a \"reporter\" gene).

1) Which method of analysis would give you the quickest way to monitor expression? Give an example of the kind of reporter gene that you could use.

2) Which method of analysis would be the most quantitative?

3) Which method of analysis would tell you if the stem-loop is a terminator/antiterminator or sequestor/anti-sequestor?

Solution

Answer:

1. The sequence 5\' CGCCGCCGCCGCCGC 3\' corresponds to the FAM probe used as a Taqman fluorescent probe.

This sequence can be replaced by the luciferase reporter reporter gene which has been widely used in monitoring the rapid expression of proteins within cells.

To judge for the expression, the vector containing the fusion protein construct can be transfected into HEK293/HeLa cells in 6 wells plate and then monitor for the expression of the proteins.

In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate and can be detected by light sensitive apparatus such as a luminometer. It is widely used because it is convenient, relatively inexpensive, and gives quantitative measurements instantaneously

2. To perform the luciferase reporter assay, the fusion construct is prepared and the resulting vector DNA are introduced into cells. The cells are allowed to grow for a period of time.

The cells are then collected , broken to release all the proteins (including the luciferase), followed by addition of luciferin and all the necessary cofactors. The measurent of the enzymatic activity is done using a luminometer. Since the gene of interest is fused to the luciferase reporter gene, the luciferase activity can be directly correlated with the activity of the protein.

The luciferase assay is extreme sensitivity and allows quantification of even small changes in transcription, and the results can be available within minutes of completing the experiment.