(2) Production of insulin - as an example of the medical use of genetic engineering in biotechnology

• The principles of genetic engineering are illustrated by the production of insulin from bacteria (shown below).

  • Genetic engineering is essentially the process of transferring a useful gene from one organism to another.

  • In this case, bacteria are genetically engineered to make human insulin.

  • The procedure uses a genetically engineered bacterium Escherichia coli and the fungus, yeast.

  • The insulin hormone is identical to that produced in the human body by the pancreas.

• 

• 1. An appropriate host bacteria is selected that will give a good yield of insulin (or any other desired product).

• 2. The bacterial plasmids are extracted from the bacteria - the plasmid acts as the vector for the insulin gene.

• 3. The vector plasmid DNA is cut by the same restriction enzymes - these enzymes recognise specific sequences of DNA and cut the DNA at these points - each end is capable of bonding with other DNA sections - hence the lovely phrase 'sticky ends' - which is are unpaired bases.

• 4. The human gene responsible for insulin production (or other genes coding for something else) is cut from the human chromosomal DNA with the same restriction enzymes - it is derived from pancreatic DNA - this cut out section of DNA also has 'sticky ends'.

• 5. From the two splits, you get reactive sites on the ends (described as 'sticky') which are short tails of unpaired bases that are complementary to each other - hence they will be able to link together.

  • Enzymes (ligases) are then added and used to insert ('splice') the insulin gene (or other desired) into the bacterial plasmid DNA, forming the recombinant DNA.

  • In other words the DNA ligase enzymes 'glue' the 'sticky' reactive ends together to reform a complete plasmid ring - this is known as recombinant DNA.

• 6. The modified plasmid vectors containing the new DNA are inserted back into the host transgenic bacteria cells.

• 7. The cloned bacteria rapidly reproduce when grown in a fermenter under highly controlled conditions - in doing so they use the inserted gene to make the protein you want e.g. in this case, the protein hormone molecule insulin.

  • So the host cells are using the inserted gene to produce the desired product e.g. insulin.

• 8. The insulin (or other product) can be produced in bulk and extracted-harvested and purified and the separated waste bacterial cells destroyed.

BUT, still one more complication!

Unfortunately, not all the host cells will have been modified correctly e.g. a faulty vector transfer.

Therefore in the final stage, you have to be able to select and identify the individual host cells that have successfully incorporated the desired gene.

Antibiotic resistance gene markers are used to identify the correctly modified host cells.

A marker gene coding for antibiotic resistance is inserted into the vector plasmid at the same time as the gene for the desired characteristic.

The host bacteria are grown in a special vessel containing antibiotics.

Only the bacteria containing the marker gene will be able to survive and reproduce, because the antibiotics will kill the rest of the cells that were not genetically modified correctly.

Working two genes in tandem! Clever stuff!

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