Question

You are given 3 tubes of RNA and asked by your supervisor to clone the gene encoding the proinsulin C-peptide that will be used later to produce insulin. The first tube contains purified mRNA isolated from pancreatic β-cells from a patient in a post-absorptive state. The second tube contains purified mRNA isolated from pancreatic α-cells from a patient during the absorptive state. The third tube contains purified mRNA isolated from pancreatic β-cells from a patient during the absorptive state. In not more than 2 pages (using 1.5 line spacing of Arial or Times New Roman fonts), provide answers for the following questions: 1) Which tube from the three is the most appropriate to use and why? 2) Describe the primary procedure (key steps, no details) that you will follow to clone the C-peptide gene from the RNA above into the only vector you have, a pUC expression vector cut open using EcoR1, which has a 5'-recognition site and a 3'-HindIII in the MCS? 3) How can you guarantee a high expression of your protein in any expression vector?

Answer 1

The most appropriate tube to use for cloning the gene encoding the proinsulin C-peptide is the first tube containing purified mRNA isolated from pancreatic β-cells from a patient in a post-absorptive state. The key steps to clone the C-peptide gene from the RNA into the pUC expression vector involve isolating the mRNA, converting it into cDNA, cutting open the pUC vector, ligating the cDNA fragment into the vector, and transforming E. coli cells with the recombinant plasmids. High expression of the protein can be guaranteed by optimizing the expression conditions.

Step-by-step explanation:

The most appropriate tube to use for cloning the gene encoding the proinsulin C-peptide is the first tube containing purified mRNA isolated from pancreatic β-cells from a patient in a post-absorptive state. This is because β-cells are responsible for producing insulin, and the post-absorptive state ensures that the cells are not influenced by the presence of nutrients that may affect gene expression.

To clone the C-peptide gene from the RNA into the pUC expression vector, the key steps would include:

1. Isolating the mRNA from the chosen tube
2. Using reverse transcriptase to convert the mRNA into complementary DNA (cDNA)
3. Adding EcoR1 restriction enzyme to the pUC expression vector to cut it open
4. Ligating the cDNA fragment into the cut pUC vector
5. Transforming E. coli cells with the recombinant plasmids carrying the C-peptide gene

High expression of the protein in any expression vector can be guaranteed by optimizing the expression conditions, such as using a strong promoter, choosing a suitable host organism, and optimizing the growth conditions for the host organism.