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 absorptive state. The third tube contains purified mRNA isolated from pancreatic β-cells from a patient during absorptive state.

Provide a response to the following questions in no more than 2 pages:
1. Which tube among the three would be the most appropriate for cloning the gene encoding the proinsulin C-peptide and why?
2. Describe the primary procedure (key steps without details) to clone the C-peptide gene from the RNA into the pUC expression vector cut open using EcoR1 and HindIII.
3. How can you ensure a high expression of your protein in any expression vector?

Answer 1

The first tube containing purified mRNA isolated from the pancreatic β-cells during a post-absorptive state would be the most appropriate for cloning the gene encoding the proinsulin C-peptide. The primary procedure to clone the C-peptide gene from the RNA into the pUC expression vector involves isolating mRNA, reverse transcribing it into cDNA, cutting open the pUC vector using EcoR1 and HindIII, and ligating the C-peptide gene into the vector. To ensure high protein expression, factors like promoter optimization, codon optimization, and growth conditions should be considered.

Step-by-step explanation:

1. The most appropriate tube for cloning the gene encoding the proinsulin C-peptide would be the first tube containing purified mRNA isolated from the pancreatic β-cells in a post-absorptive state. This is because the proinsulin C-peptide is primarily produced in the pancreatic β-cells and its expression is expected to be higher in these cells during a post-absorptive state.

2. The primary procedure to clone the C-peptide gene from the RNA into the pUC expression vector cut open using EcoR1 and HindIII involves the following key steps:

• Isolate mRNA from the desired tube containing the gene of interest (β-cells from post-absorptive state).
• Reverse transcribe the mRNA into complementary DNA (cDNA) using reverse transcriptase.
• Generate double-stranded cDNA using DNA polymerase-I.
• Use restriction enzymes (EcoR1 and HindIII) to cut open the pUC expression vector.
• Ligate the C-peptide gene cDNA into the cut open pUC vector.
• Transform the recombinant plasmids into E. coli cells for propagation and expression.

3. To ensure a high expression of the protein in any expression vector, several factors should be considered such as optimizing the promoter used in the vector, codon optimization for the host organism, using strong ribosomal binding sites, and optimizing growth conditions.