Insulin Production - GM case study

What is Insulin?

• A hormone made up of a polypeptide of 51 amino acids
• Produced in the pancreas
• Converts glucose in the blood to gylcogen and stores it in the liver
• It was discovered before the human genome was mapped

Why?

• To treat Type 1 diabetes (melitus) where the pancreas does not produce enough insulin or metabolic disease. (In type 2 diabetes, the body cell's are immune to insulin)

Insulin for clinical use was extracted from pancreatic tissue of slaughtered pigs. Problems: not identical to human tissue, expensive, less effective and only a small amount of insulin is present

• Use specialised centrifugation methods to separate the mRA of the right length from pancreati tissue
• once found, reverse transcriptase is used to synthesis a complementary strand
• Template strand of DNA is formed
• DNA polymerase is added with free nucleotides so that a second strand is formed - a copy of the original gene is produced (cDNA)
• Unpaired nucletodes are added at each end of the gene to give ticky ends complimentary to those on the cut plasmid
• Plasmids are then cut open using restriction enzymes and mixed with cDNA so some plasmids take up the gene
• DNA ligase seals up the plamsids to form recombinant plasmids
• The plasmids are then mixed with bacteria, some of which take p the plasmids
• The bacteria are then grown on an agar plate and they replicate so some bacterial cells now produce human insulin

1) Bacteria that did not take up a plasmid

2) Bacteria that have taken up a plasmid that did not take up the insulin gene and sealed to form the recombinant plasmid

3) Transformed Bacteria - bacteria that have taken up the recombinant plasmids

Replica Plating

• Identification of transformed bacteria as the original plasmids have a resistance gene to two antibacterial chemicals (ampicillin and tetracycline) - the genetic markers
• The bacteria originally are susceptible to both of those

The plasmids are cut by a restriction enzyme that has a target site in the middle of the tetracycline resistance gene , so that if the insulin gene is taken up, the gene for tetracycline resistance is broken up and does not work. The gene for ampicillin resistance does work

• Some colonies are transferred onto agar that has been made with ampicillin, so only those that have taken up a plasmid will grow
• Some cells from these colonies  are then transferred onto agar that has been made with tetracycline so only those that have taken up a plasmid without insulin will grow
• By tracking these colonies, we know that any bacteria tha grow on ampicillin, but not on tetracycline, have taken up the plasmid with the insulin gene

We can now identify the colonies we want and grow them on a large scale. These bacteria then produce insulin on a large scale which can be harvested for use