Aulanni

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Genetic Engineering

• Aulanniam
• Biochemistry Laboratory
• Chemistry Department
• Brawijaya University

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First, the nucleus of human cells are burst
Human cell
Nucleus
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The chromosomes are cut up into small fragments
and the required gene identified.
Fragment containing required gene
Chromosome fragments
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Cytoplasm
Plasmid
Bacterial cell wall
Bacterial chromosome
Structure of a typical bacterium
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Genetic Engineering
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 theGenetic engineer.
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Genetic Engineering
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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Genetic Engineering
P
Cut here
T
By using special enzymes, we can make a cut in
the midst of ONE of theseantibiotic resistance
genes.In this example, we will cut open the T
gene
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Genetic Engineering
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
plasmidso that the green T gene will no longer
work correctly.
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Genetic Engineering
As plasmids are extremely small, we cannot tell
by looking which ones have gotthe human gene in
the right place. We need to use a shotgun
approach andincubate thousands of plasmids with
hundreds of bacterial cells
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Genetic Engineering
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
antibioticresistance genes.
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Genetic Engineering
Agar containingpenicillin
An agar plate containing Penicillin is used to
allow only those cells which havetaken up a
suitable plasmid to survive and divide. These
cells must have resistanceto 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 WONT grow in the
presence of Tetracycline
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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
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to be continued...