Engineering insulin

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  • Created by: Steff06
  • Created on: 05-06-16 15:31
What condition do people who cannot produce the hormone insulin suffer from?
Type 1 diabetes mellitus
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What has previously been used for insulin?
Insulin used to be extracted from the pancreatic tissue of pigs.
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What were the problems of using insulin from pigs?
It is less effective and very expensive as only a small amount is present in the tissue.
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As the DNA code for insulin is small what did scientists try to find instead and how?
Tried to find the mRNA for the gene and separated the mRNA from the pancreatic tissue using centrifugation methods.
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What did they do once they had found the mRNA?
Enzyme reverse transcriptase was used to synthesise a complementary DNA strand.
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How is the second strand built on using the copied DNA as a template?
By adding DNA polymerase and a supply of DNA nucleotides.
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What does this produce and what are added?
Produces a copy of of the original gene called cDNA. Unpaired nucleotides are added to each end to give complementary sticky ends.
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How are the plasmids cut open and what are they mixed with?
Cut open with a restriction enzyme and mixed with cDNA genes.
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What happens if a plasmid takes up the gene?
DNA ligase enzyme seals up the plasmid which are called recombinant plasmidsas they contain a new piece of DNA.
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What then happens to these plasmids?
The plasmids are mixed with bacteria and some take up the recombinant plasmids.
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Where is the bacteria placed?
Grown on an agar plate where each bacterial cell grows to produce a mound of identical cells called a colony.
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What are the 3 possible types of colony that may grow?
Some from bacteria without a plasmid. Some from bacteria with a plasmid but without the gene. Some that have taken up the recombinant plasmid with the gene - we want these.
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What is the modern process used to identify transformed bacteria?
Replica plating and using genetic markers.
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What are chosen as the genetic markers?
The original plasmids as they carry the genes that make any bacteria receiving them resistant to ampicillin and tetracycline.
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What is susceptible to both of the antibiotic chemicals?
The bacteria such as E.coli.
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How are the plasmids then cut and why?
Plasmids cut by a restriction enzyme with its target site in the middle of the tetracycline resistance gene/
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Why are the plasmids cut like this?
So if the required gene is taken up, the gene for tetracycline resistance is broken up and doesn't work but the one for ampicillin resistance still works.
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What is the 1st step of replica plating?
Bacteria are grown on standard nutrient agar so all bacterial cells grow to form colonies.
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What happens to some cells from the colonies?
Some transferred to agar made with ampicillin so only those that took up a plasmid will grow.
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Where will some other cells from the colonies be transferred to?
Some transferred to agar made with tetracycline so only those with the plasmid without the insulin gene will grow.
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How do we know which have the insulin gene?
Bacteria grown on the ampicillin and not tetraycline must have the plasmid with the insulin gene.
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Describe the reverse transcriptase enzyme
Enzyme copies viral RNA back to form DNA which is transcription in reverse.
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Other cards in this set

Card 2

Front

What has previously been used for insulin?

Back

Insulin used to be extracted from the pancreatic tissue of pigs.

Card 3

Front

What were the problems of using insulin from pigs?

Back

Preview of the front of card 3

Card 4

Front

As the DNA code for insulin is small what did scientists try to find instead and how?

Back

Preview of the front of card 4

Card 5

Front

What did they do once they had found the mRNA?

Back

Preview of the front of card 5
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