Manipulating genomes- insulin Flashcards
how is mRNA extracted for human insulin production? FIRST STEP OF INSULIN PRODUCTION
-Rather than try and find the relatively small insulin gene amongst the whole genome, the mRNA is extracted instead from the cytoplasm of human β cells, after transcription
-It is separated from other cell components and other mRNA with a centrifuge
How is cDNA synthesised (complementary DNA) from mRNA? SECOND STEP OF INSULIN PRODUCTION
The mRNA is then treated with reverse transcriptase enzyme, turning it onto a single strand of DNA, complementary to the original- cDNA
What occurs after cDNA is synthesised? THIRD STEP OF INSULIN PRODUCTION
-Free DNA nucleotides are added along with DNA polymerase
-This builds a complementary strand alongside the template
-This double stranded copy of the original gene is called a cDNA gene
FORTH STEP OF INSULIN PRODUCTION?
Unpaired nucleotides are then added, giving sticky ends of a particular sequence (they will be complementary to the cut plasmid in the next step)
FIFTH STEP OF INSULIN PRODUCTION?
-Plasmids are removed from E.coli bacteria and cut open at a specific site using a restriction enzyme
-This will leave sticky ends complementary to those from the human insulin cDNA
SIXTH STEP OF INSULIN PRODUCTION?
-The plasmids are mixed with the cDNA genes in the presence of DNA ligase enzyme
-This reseals the plasmid, with some of them incorporating the human insulin gene
-These are now called recombinant plasmids
SEVENTH STEP OF INSULIN?
The plasmids are mixed with the E.coli bacteria and heat shocked with calcium salts to cause them to take up the plasmids
EIGHTH STEP OF INSULIN?
The next stage is to identify which bacteria have taken up the recombinant plasmid, by replica plating
What are the three possible types of bacteria which can be formed by replica plating?
-Ones that have not taken up a plasmid at all GROUP A
-Ones that have taken up a plasmid which re-sealed itself, without incorporating the human insulin gene GROUP B
-Ones which did take up a recombinant plasmid. These are called transformed bacteria, and are the ones we want GROUP C
Plasmid contains two genes for resistance against what antibiotics?
ampicillin and tetracycline
where does the restriction enzyme cut the plasmid?
in the middle of the tetracycline resistant gene. Therefore this means, if the human insulin gene is successfully added, then tetracycline resistant gene will no longer work
How is the recombinant bacteria collected?
-collection of bacteria are grown on nutrient agar which contains ampicillin. This kills any bacteria which did not take up the plasmid.
-replica plates are then formed by transferring some of the cells from each colony onto a new agar plate containing tetracycline. Any cells which grow are from group B and colonies which failed to grow are from group C.
-transformed bacteria can then be removed and grown on a large scale. The insulin which they produce is collected and purified.
Describe golden rice
-rice is being genetically engineered to contain vitamin A as many people face vitamin A deficiency causing blindness
