Gene Markers

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How do we identify bacteria that have taken up the plasmid?
We use the fact that over many years some bacteria have developed resistance against antibiotics.
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How do most bacteria resist the affect of antibiotics?
By producing an enzyme that breaks down the cell wall of the bacterium before it can attack it.
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Where are the genes stored for antibiotics resistance?
In the plasmid.
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What is the first step of using antibiotic resistance markers?
Bacterial cells are grown on a medium of ampicillin. bacterial cells that have taken up the plasmid will have antibiotic resistance and so will survive, the ones that haven't taken up the plasmid will not be resistant and will die.
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Why are gene markers used?
They involve using a second separate gene that is on the plasmid.
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Why is the second gene used by the gene marker often identifiable? (3 reasons)
1. It codes for antibiotic resistance. 2. It codes for a fluorescent protein that can be visibly seen. 3. It may produce an enzyme whose action can be identified.
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What is replica plating?
This uses the OTHER antibiotic resistant gene that was cut to incorporate the DNA fragment. As this gene is cut it will no longer produce resistance to that antibiotic.
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Replica plating is..
Bacterial cells that survive the 1st treatment with antibiotics are known to have taken up the plasmids. They are cultured by being spread very thinly on an agar plate. A tiny sample of each colony is transferred to a 'replica' plate.
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What does the replica plate contain?
The second antibiotic against which the antiobiotic resistance gene will have been made useless if the new gene has been taken up.
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How will we (at the end stage) identify which bacteria have taken up the plasmid?
the ones that die from the second antibiotic have taken up the plasmid. We then look at their location on the second replica plate as they are in the same position. We know that these are the useful bacteria.
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Explain the use of fluorescent markers?
Transferal of a gene from a jellyfish into the plasmid. The gene fluoresces. The gene REQUIRED is transported to the centre of GFP. So if a bacterial cell has taken up the gene it WILL NOT fluoresce.
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Why is the use of fluorescent markers helpful?
Because it is very rapid.
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What is an example of an enzyme marker?
The enzyme LACTASE.
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How is the enzyme Lactase used as a gene marker?
Lactase will turn a particular colourless substrate blue. The required gene is transported into the gene that makes LACTASE. Therefore, if the plasmid does not turn a substrate blue then the plasmid HAS been taken up.
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Card 2

Front

How do most bacteria resist the affect of antibiotics?

Back

By producing an enzyme that breaks down the cell wall of the bacterium before it can attack it.

Card 3

Front

Where are the genes stored for antibiotics resistance?

Back

Preview of the front of card 3

Card 4

Front

What is the first step of using antibiotic resistance markers?

Back

Preview of the front of card 4

Card 5

Front

Why are gene markers used?

Back

Preview of the front of card 5
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