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# Production of Human Insulin using Escherichia coli **Question 46** Explain in detail the production of human insulin using *Escherichia coli*. (15 marks) **Answer:** 1. **mRNA Extraction:** mRNA containing genetic codons for insulin synthesis is extracted from the β-cells of the pancreatic isle...

# Production of Human Insulin using Escherichia coli **Question 46** Explain in detail the production of human insulin using *Escherichia coli*. (15 marks) **Answer:** 1. **mRNA Extraction:** mRNA containing genetic codons for insulin synthesis is extracted from the β-cells of the pancreatic islet of Langerhans. 2. **cDNA Synthesis:** The extracted mRNA is placed in a medium with reverse transcriptase and free nucleotides to synthesize single-stranded complementary DNA (cDNA). 3. **cDNA Doubling:** The single-stranded cDNA is doubled to produce double-stranded cDNA, catalyzed by DNA polymerase. 4. **Cloning Vector:** A bacterial plasmid containing the *amp* gene (for ampicillin resistance) and the *lac Z* gene (used as a marker) is used as the cloning vector. 5. **Digestion:** Both the cDNA fragment containing the insulin gene and the bacterial plasmid are cut using the same restriction enzyme, producing sticky ends. The bacterial plasmid is cut at the *lac Z* gene. 6. **Insertion:** The DNA fragment carrying the insulin gene is inserted into the opened plasmid using DNA ligase. 7. **Recombinant DNA:** The insertion creates recombinant DNA (containing the insulin gene). 8. **Transformation:** The recombinant DNA is introduced into host cells (*E. coli*) using chemicals such as Ca2+ ions or weak electrical pulses to form pores in the bacterial membrane, allowing plasmid entry. 9. **Screening:** Bacteria are screened for those containing the cloned insulin gene using nutrient agar and X-gal (a substrate). 10. **Colony Color:** * Bacteria with a functional *lac Z* gene produce blue colonies. * Bacteria having recombinant DNA with a non-functional *lac Z* gene produce white colonies. 11. **Amplification:** Bacteria containing the cloned insulin gene are isolated and cultured in a fermenter vessel for amplification. 12. **Harvesting and Purification:** Cloned cells are removed from fermenter and lysed, followed by insulin extraction and purification in large quantities.