Insulin Production - Process Flashcards
What is the first step in Insulin Production?
What is extracted and cut?
- Plasmids are extracted from Bacteria
- Gene of interest - insulin - is cut out from humans using a restriction enzyme to cut sticky ends
- Same restriction enzyme is used to cut up plasmid
- They are placed in a test tube solution
What is the 2nd step in Insulin Production?
Formation of certain plasmids
- DNA Ligase in test tube joins together complementary sticky ends by reforming phosphate-diester bonds.
- Some plasmids close-back up without gene of interest
- Some plasmids take up target DNA (gene of interest)
What is the 3rd step in Insulin Production?
What plasmids are formed
- Plasmids without gene of interest are called non-recombinant plasmids
- Plasmids with gene of interest are called recombinant plasmids
What is 4th step in Insulin Production?
Bacteria
- Plasmids are put in same test-tube as bacteria
- Heat Shock is used where test-tube is originally in ice-bath
- Then suddenly moved to 42 degrees celsius
- Heat causes increased fluidity in bacterial membrane, increasing permeability
- Some bacteria take in plasmids
Could talk about electroporation instead
What is 5th step in Insulin Production?
Types of bacteria formed
- Bacteria that contain plasmids are called transformed bacteria
- Bacteria without plasmids are called untransformed bacteria
What is 6th step in Insulin Production?
testing for transformed bacteria
- All bacteria are first plated on an ampicilin agar plate.
- Transformed bacteria will survive untransformed will not.
- Transformed bacteria will live as plasmids contain ampicilin resistance gene
What is 7th step in Insulin Production?
Testing for recombinant plasmids
- Surviving colonies are taken and plated on tetracycline
- Non-resistant bacteria contain recombinant plasmids
- Resistant bacteria contain non-recombinant plasmids
- Recombinant plasmids have tetracycline resistance gene interrupted by insulin gene
What is 8th step in Insulin Production?
What happens to recombinant transformed bacteria?
- Plasmids once again extracted
- Using same restriction ezyme are cut
- lacZ gene is inserted adjacent to insulin gene
- Some plasmids will take in lacZ others won’t, and are ligated by Ligase
lacZ’s stop codon is not included
What is 9th step in Insulin Production?
What happens to new plasmids with
- Same process of heat/shock or electropration is conducted again
Refer to steps 3-5 for more detail
What is 10th step in Insulin Production?
Testing for transformed bacteria with lacZ gene
- Bacteria are plated on ampicilin and X-gal agar plate
- Surviving colonies are all transformed bacteria
- Colonies that are blue contain recombinant plasmids with both insulin and lacZ gene
- The B-galactosidase produced by lacZ gene converts colourless X-gal product to blue
What is 11th step in Insulin Production?
What is done to final desired bacterial colonies?
- Desired tranformed recombinant plasmid containg bacteria are placed in conditions for reproduction
- They sythesise fusion protein of B-galactosidase and insulin subunit
What is 12th step in Insulin Production
What is done to replicated bacteria
- Bacterial membranes are broken and Cyanogen Bromide is added to break apart B-galactosidase and insulin subunit
- First insulin sub-unit is produced
Don’t need to remember cyanogen bromide
What is 13th step in Insulin Production?
What is done for second insulin subunit?
Who process in repeated, except using second insulin gene as gene of interest
What is 14th step in Insulin Production?
How are insulin subunits joined?
- Both insulin sub-units are mixed
- Disulphide bonds are formed (automatically) between each insulin sub-unit
- Final quarternary functional insulin is produced!
Yay
