Transcript Genetic Engineering Ppt

Genetic Engineering
First, the nucleus of human cells are burst
Nucleus
Human cell
Genetic Engineering
The chromosomes are cut up into small fragments and the
required gene identified.
Fragment containing required
gene
Chromosome
fragments
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Next the fragments are spread out and the required one
isolated.
Segment with required gene
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Cytoplasm
Bacterial cell wall
Plasmid
Bacterial chromosome
Structure of a typical bacterium
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Plasmid
Plasmids are loops of DNA separate from the main chromosome. They carry
genes for things like antibiotic resistance. This makes them very useful to the
Genetic engineer.
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P
T
In the above plasmid, the YELLOW gene is one that gives the bacterium
resistance to one antibiotic (eg Penicillin).
The GREEN gene gives
resistance to a different antibiotic (eg Tetracycline)
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P
Cut here
T
By using special enzymes, we can make a cut in the midst of ONE of these
antibiotic resistance genes.In this example, we will cut open the ‘T’ gene
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Prepared
human gene
Next, we introduce the prepared HUMAN gene to the mixture.
If all goes according to plan, the human gene will fit into the cut in the plasmid
so that the green ‘T’ gene will no longer work correctly.
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Intact P gene and
‘defective’ T gene
P and T
Genes intact
No P or T
gene
As plasmids are extremely small, we cannot tell by looking which ones have got
the human gene in the right place. We need to use a ‘shotgun’ approach and
incubate thousands of plasmids with hundreds of bacterial cells
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Required cell
Cell with P and T intact
Cell with neither P or T
Some cells will take up the recombinant plasmid, some will take up original
plasmids, others will take up no plasmds at all or ones without antibiotic
resistance genes.
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Agar containing
penicillin
Colonies growing from
single cells that are
resistant to penicillin
An agar plate containing Penicillin is used to allow only those cells which have
taken up a suitable plasmid to survive and divide. These cells must have resistance
to Penicillin
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Next, these colonies are sub-cultured onto agar containing tetracycline.
Only cells resistant to BOTH antibiotics will be able to grow.
We are interested in those cells which WON’T grow in the presence of
Tetracycline
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These cells must
have intact T genes
These cells must
have intact P genes
and defective T
genes
Next, these colonies are sub-cultured onto agar containing tetracycline.
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This colony will probably have the correct plasmid to produce the product from the
human gene. Cells from this colony will be grown on a large scale and the medium
analysed for the presence of the product from the human gene, eg growth hormone