Human Insulin Production with E. coli

The mRNA that codes for the synthesis of insulin is extracted from the β-cells of the islets of Langerhans in the pancreas. This mRNA is then placed in a medium containing reverse transcriptase and free nucleotides to synthesize single-stranded complementary DNA (cDNA). The single-stranded cDNA is doubled to produce double-stranded cDNA, catalysed by DNA polymerase. The cDNA fragments with the insulin gene and lacZ gene are used as the cloning vector. The bacterial plasmid is cut at the lacZ gene and the bacterial plasmids are cut with the same restriction enzyme to produce sticky ends. The DNA fragment with the insulin gene is inserted into the opened plasmid. The sticky ends allow the opened plasmid and the DNA fragment to anneal/splice with the help of DNA ligase. The recombinant DNA with the insulin gene is produced. The recombinant DNA is transformed into the host cell, E. coli. Transformation is done using chemicals such as Ca²⁺ ions or weak electrical pulses to facilitate the process (by forming pores in the bacterial membrane to allow the entry of the plasmid). The bacteria are screened for organisms with the cloned genes, in which the bacterial colonies are cultivated in a medium containing the antibiotic ampicillin and X-gal sugar. Bacteria with functional lacZ gene produce blue colonies. Bacteria with non-functional lacZ gene produce white colonies. The bacteria with the cloned genes are isolated and cultured in a suitable fermenter vessel for amplification. The cloned cells are removed from the fermenter, and then they are lysed. Insulin is extracted and purified in large quantities.