A hypothetical protien call GAC was found to inhibit the mitosis promoting facto

Question

A hypothetical protien call GAC was found to inhibit the mitosis promoting factor, and has shown promise as a cancer therapy. This protien needs to be made in large quanities in a yeast cell then tested for function. You are given the:

1. Sequence of the gene and

2. The promoter and regulator sequences.

Draw a concept map (can be shown in listed steps) to show every single step you will take to make this protien in a yeast cell and then test the protien and its effect on the cell cycle (inhibits mitosis promoting factor).

***please include detailed steps for DNA transcription/translation and how the RNA is made to the protien (include how the promoter and regulator sequences would be used) and then added to the cell and how it would be tested as an inhibitor of the mitosis-promoting factor.   *** Please be thorough, I will rate 5 stars! Thank you!!!

Explanation / Answer

If the sequence is given, make primers for the gene for a PCR. Once we get the gene specific primers, make many copies of the gene using PCR. Insert restriction sequences in the primers before ordering so that after PCR, we can use some restriction enzymes to cut the product and clone it in to a plasmid. Use same restriction enzymes for both plasmind and the gene so that annealing would be easy. Once annealed, we clone them in to yeast. Choosing plasmid is an important step. Make sure that plasminds have the promoter and regulator sequences in it. Once cloned, incubate yeast at a proper temperature with all the nutrients to make them divide happily. To measure the effect of the cloned gene on yeast cell cycle, we can directly isolate proteins from yeast and measure on westernblot and look for cell cycle markers. If the markers are not expressed or less expressed, it means that, the gene has an effect on yeast cell cycle. You can also measure the inhibition of mitosis by using radio active materials in a thymidine incorporation assay etc.

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