U3 AOS1 CH3 PART 2/B VCE BIO insulin

whats insulin and type 1 diabetes

peptide hormone that promotes uptake of sugar from bloodstream and storage in muscle or fat tissue.

essential for normal function, w/o insulin the body relies on fat as energy source

• lead to build up of dangerous substances in the blood

type 1 diabetes an auto-immune disease in which insulin producing cells within the immune system are attacked by the bodies own immune system, unable to produce sufficient insulin.

• people have to inject themselves with insulin to maintain normal blood glucose levels.

bacteria & copies

• no introns as bacteria can not splice them out of mRNA

• mRNA converted back into dna → cDNA, and are put through PCR to make lots of copies of human insulin gene

steps to making recombinant insulin

1. insulin protein made in two parts (joined in final steps).

   • insulin chain a/b extracted from cell, introns removed, exons spliced together

2. plasmids removed from bacteria and restriction enzymes ecoRI and bamHI cut plasmid.

   - same restriction enzyme cuts insulin gene and b-gal gene (has no stop codon so it can transcribe all together) to have all genes complementary overhands and join together with plasmid, using ligase

3. DNA litigation doesnt always work. plasmids either

   • reseal themselves, only take up insulin gene, take up both insulin and b-gal gene

   plasmids are mixed with bacteria and has electrical shock to make bacteria membrane permeable to take plasmids. (transformed bacteria with recombinant plasmid)

4. bacteria are screened on a plate with antibiotics and x-gal

   Bacteria w/o plasmid die → dont have antibiotic resistance gene

   bacteria w plasmid live → has antibiotic resistance gene from b-gal, b-gal makes protein to activate with x-gal = blue bacteria

   • white bacteria dont have b-gal gene

5. extracted blue bacteria grown into large colony, bacteria transcribes the gene for insulin producing mRNA for insulin chain, the mRNA chain undergoes translation at ribosome producing insulin chain.

6. insulin a and b extracted from diff bacteria cell by rupturing cell wall.

   insulin chains purified → removing b-gal from both chains

   insulin chains a and b combine together = disulphide bonds

FUNCTIONAL RECOMBINANT PLASMID PROTEIN FOR INSULIN

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