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.

Which tube from the three is the most appropriate to use and why?
a) Tube 1, as it contains purified mRNA isolated from pancreatic β-cells in a post-absorptive state.
b) Tube 2, as it contains purified mRNA isolated from pancreatic α-cells in an absorptive state.
c) Tube 3, as it contains purified mRNA isolated from pancreatic β-cells in an absorptive state.
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.
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 Tube 1, as it contains purified mRNA from pancreatic β-cells. The primary procedure for cloning the gene involves reverse transcription, amplification, cloning into a vector, transformation, and verification by sequencing. To ensure high protein expression, factors such as promoter strength, codon optimization, and fusion tags can be considered.

Step-by-step explanation:

To clone the gene encoding the proinsulin C-peptide from the given RNA samples, the most appropriate tube to use is Tube 1, as it contains purified mRNA isolated from pancreatic β-cells in a post-absorptive state. This is because the proinsulin C-peptide is mainly produced in the pancreatic β-cells. To clone the gene, the following primary procedure can be followed:

1. Isolate the RNA from the chosen tube.
2. Reverse transcribe the mRNA into cDNA using reverse transcriptase.
3. Amplify the cDNA using PCR with primers specific to the proinsulin C-peptide gene.
4. Clone the amplified cDNA into the pUC expression vector that has been cut open using EcoR1 and HindIII restriction enzymes.
5. Transform the recombinant plasmid into E. coli cells and select for transformed cells.
6. Verify the presence of the cloned gene by sequencing.

To guarantee a high expression of the protein in any expression vector, it is important to consider factors such as using a strong promoter, optimizing codon usage for the host organism, and considering the use of fusion tags or signal sequences to enhance protein expression and stability.