Production of Humulin by recombinant E. coliI.

Finding the insulin gene

Method 1: Using reverse transcriptase to synthesise cDNA.
Extract and purify mRNA from Beta cells from the islets of Langerhans. Incubate mRNA with reverse transcriptase and activated deoxyribonucleotides. Remove original mRNA strand using RNAase or alkali to produce a single stranded cDNA copy. Complete the double stranded DNA molecule using DNA polymerase and activated deoxyribonucleotides. Sticky ends are added.

Finding the insulin gene

Method 2: Using chemical synthesis of DNA molecule using the base sequence gleaned from the primary structure of the two polypeptides.
Synthesise the 63 and 90 polynucleotide chains coding for chain A and B of insulin. Add a stop codon at the 3 end. At the 5 end add methionine anticodon. Sticky ends are added.

Finding the insulin gene

Method 3: Make a genomic library of the DNA.
Extract all DNA from beta cell and perform a partial digest. This involves using a restriction endonuclease (e.g. SAU 1A) and cutting the genetic information into smaller pieces. These DNA fragments are inserted into a vector and cloned to form a genomic library. The library can then be screened using a radioactive probe for the insulin genes and the genes cut out using restriction endonucleases, producing sticky ends.

Inserting the gene into a vector (The plasmid).

Plasmid is a small circular piece of DNA. They can contain genes which confer antibiotic resistance. To insert gene the plasmid is cut open using a restriction endonuclease, leaving a short length of unpaired bases at each end (sticky ends). The DNA is inserted using the sticky ends complementary to those on the plasmid and the phosphodiester bond formed using an enzyme called DNA ligase. This is called recombinant DNA.

Inserting the gene into a vector (The plasmid).

Inserting the vector into the required organism (E. coli).

The recombinant plasmid is inserted into the bacteria by the process of transformation. The recombinant bacteria are sorted by growing them in the presence of an antibiotic. The bacteria which survive are the ones which have taken up the plasmid. They are said to be transformed.

Culturing recombinant E. coli and insulin synthesis.

These recombinant bacteria are then produced in a monoculture and the two insulin chains are synthesised by the recombinant bacteria. The two polypeptides accumulate in the fermentation liquor and are extracted and purified. Insulin is finally formed when the two polypeptide chains are joined using disulphide bonds.

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