Genetic Engineering

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 Chromosome

gene 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 Plasmid

Bacterial cell wall

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

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

No P or T
gene
P and T
Genes intact

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