Gene Transfer- inulin production Flashcards
what is a vector in terms of insulin production? what is the host cell?
-a bacterial plasmid
-bacteria cell
what are the 7 steps involved in insulin production?
-isolation of required gene that codes for the protein
-cutting the gene out of its DNA
-preparation of target DNA (cut it out)
-insert insulin gene into plasmid
-insert plasmid back into bacteria cell
-identify target/ transformed cells
-culturing host cells
how is the DNA placed into plasmids, what must be used?
-as the bacterial plasmid is a closed loop it has to be cut with a restriction enzyme
-if the same restriction enzyme is used to cut out the section of DNA it will mean that the plasmid and donor DNA will have complementary sticky ends
what is the enzyme used to fuse the donor DNA and the plasmid together?
-DNA ligase anneals the gene to the complementary sticky ends on the plasmid
what happens to the shape of the plasmid once the human gene is inserted?
-the plasmid becomes a closed loop again
what is the plasmid referred to as once it contains the human insulin gene?
-recombinant plasmid
what is another type of cell that can be used as a vector? how can it?
-a bacteriophage virus which can have donor DNA spliced into it
-due to viruses being adapted to shoot their genetic material in a bacteria cell, the modified virus can do this too as shoot out donor DNA also
what is the next step after the plasmid takes up the insulin gene?
-encourage the host cell (bacteria) to take up the plasmid
why is bacteria used as the host cell?
-they can produce large amounts of plasmids in a short period of time
-divide quickly too
what are the ways that bacteria cells are encouraged to take up the modified plasmids?
-bacteria cells can be incubated with calcium ions and subjected to heat shock
what is the purpose of using heat shock for the bacteria cells?
-it makes the bacteria more permeable
-reduces the barrier for bacteria cells to take up the plasmids
what is the next step after the plasmid is inserted?
-identify the transformed bacteria (bacteria that contains the donor DNA)
what are marker genes? what is an example of a marker gene used in insulin production?
-other genes on the recombinant plasmid apart from the donor DNA
-R-plasmids
what are marker genes used for?
-used to identify the transformed bacteria
what do most plasmids carry a gene for? what is this specific gene in the R plasmid?
-most plasmids carry the gene for antibiotic resistance
-tetracycline and ampicillin
what happens to the genes for tetracycline and ampicillin if a piece of donor DNA is inserted into the plasmid?
-if the donor DNA anneals effectively into the plasmid (recombinant plasmid is produced) the plasmid will no longer have the gene that allows resistance to tetracycline
-it has been interrupted by the donor DNA
-however the gene that codes for resistance to ampicillin will remain unaffected
how is the R plasmid tested to see if the donor DNA has been taken up?
- the bacteria, with the gene that is resistant to tetracycline, are encouraged to take up the recombinant plasmids
-the bacteria is transferred to an agar plate with ampicillin and colonies of the bacteria are left to develop
-colonies will only develop from bacterial cells that are resistant to ampicillin
-they will have taken up the plasmid, not necessarily the plasmid with the donor DNA
-a replica plate is made and bacteria is transferred to an agar plate with tetracycline and the colonies are left to develop
-this plate will be missing some of the colonies that are present on the plate containing the ampicillin
-the missing ones will have taken up the recombinant plasmids, as they are no longer resistant to tetracycline as that gene has been disrupted
how does replica plating occur? what is the key thing about this?
-it involves blotting the original plate with an absorbing pad and then pressing it against the surface of a fresh agar plate
-the key this is that the colonies will or wont form on equivalent positions on the new plate
what is the last stage of the insulin production?
-once it has been confirmed that the colony of bacteria contains the recombinant DNA the next and last phase is to clone the bacteria in large fermenters
what is the term used to describe the transformed bacteria?
-genetically modified organism
