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. In not more than 2 pages (using 1.5 line space of Arial or Times New Roman fonts) provide answers for the following questions? Which tube from the three is the most appropriate to use and why?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?
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. In not more than 2 pages (using 1.5 line space of Arial or Times New Roman fonts) provide answers for the following questions? Which tube from the three is the most appropriate to use and why?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?
Insulin is a peptide hormone which is released by beta cells of pancreatic islets of Langerhans. It is composed of two polypeptide chains, alpha and beta. The two chains are joined together by disulphide bridges. The insulin hormone regulates the metabolism of carbohydrates, fats and protein by promoting the absorption of glucose from blood and its storage in adipose tissue and muscle cells in the form of glycogen. Synthesis and secretion of insulin in insufficient amounts causes a person to suffer from diabetes mellitus.
Proinsulin is a single peptide chain, precursor to insulin, which is produced on the ribosomes of beta cells. They are the immature form of insulin which undergoes maturation in absorptive stage.
Absorptive stage is the digestion and absorption state, when the ingested food causes glucose levels to rise in the blood. The pancreatic cells release insulin in response to high blood sugar levels.
Post absorptive is the phase when the level of glucose in blood is low and all the available glucose has been utilized by the body. The production of insulin stops and glucagon secretion starts such as to convert stored glycogen to glucose. Glucose is then utilized by the body for energy requirements.
1) Among the three test tubes provided, the last tube containing mRNA (messenger ribonucleic acid) isolated from the patient in the absorptive stage is most appropriate for cloning of the gene.
- In post absorptive state glucagon is produced, which is antagonistic to insulin. Therefore, insulin is not produced. Hence, the first tube is not an appropriate sample for cloning of proinsulin.
- The second tube will not be used as insulin is secreted by beta cells and not alpha cells.
- The insulin is produced by beta cells and the secretion of insulin is higher in absorptive state due to high levels of glucose. Therefore, the third tube will be used for cloning.
2) Plasmid is the extrachromosomal material present in the bacteria which is self-replicating. Plasmids are mainly used as vectors for transferring genes of interest to the host.the key features of a plasmid vectors are, origin of replication, multiple cloning site, selectable marker and high copy number.
Restriction enzymes also called molecular scissors which creates nicks in double stranded DNA of either the gene of interest of the plasmid DNA to create overhangs for ligation.
Procedure of cloning proinsulin gene:
- The purified mRNA extracted from the beta cells contains a small compartment coding for the proinsulin gene.
- During the process of purification, the mRNA is bound to oligo (dT) cellulose beads.
- The mRNA is further purified by gel electrophoresis
- The mRNA bound with oligo dT(deoxy thymidine) tail is utilized as a template for the cDNA (complementary DNA) synthesis.
- Reverse transcriptase enzyme synthesizes DNA (deoxyribonucleic acid) strand complementary to the mRNA with the help of primer complementary to the 3” end. Here primer dG-dC (deoxy guanine-cytosine) is used.
- RNA-DNA heteroduplex is formed.
- RNAse H cuts the hair pin loop separating the mRNA and DNA.
- DNA polymerase synthesizes another DNA strand complementary to the DNA template thereby copying the mRNA.
- The pUC plasmid is spliced by an EcoR1 (Escherichia coli strain R1)restriction enzyme creating sticky ends at 5’ and a logo nucleotide dG-dC is attached
- The cDNA (complementary DNA) spliced by the same restriction enzyme.
- Both the plasmid and the cDNA have complementary ends which are then ligated by DNA ligase.
- The recombinant plasmid is then transferred to the host (E.coli) Escherichia coli
- Screening of the recombinant plasmid is done in the media containing antibiotic ampicillin or tetracycline.
- The cells containing recombinant plasmid were able to grow in antibiotic medium as the gene coding for ampicillin or tetracyclic resistance is expressed.
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