2/14/2012

Genetic Engineering
First, the nucleus of human cells are burst

Genetic Engineering
The chromosomes are cut up into small fragments and the required gene identified. Human cell
Fragment containing required gene Chromosome fragments

Nucleus

Genetic Engineering
Next the fragments are spread out and the required one isolated.

Genetic Engineering
Cytoplasm Plasmid

Bacterial cell wall
Segment with required gene

Bacterial chromosome

Structure of a typical bacterium

Genetic Engineering
Plasmid

Genetic Engineering
P

T
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. 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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Only cells resistant to BOTH antibiotics will be able to grow. we cannot tell by looking which ones have got the human gene in the right place. we introduce the prepared HUMAN gene to the mixture.In this example. some will take up original plasmids. we will cut open the µT¶ gene Genetic Engineering Intact P gene and µdefective¶ T gene defective¶ Genetic Engineering P and T Genes intact No P or T gene Required cell Cell with P and T intact Cell with neither P or T As plasmids are extremely small. These cells must have resistance to Penicillin Next. T By using special enzymes.2/14/2012 Genetic Engineering P Cut here Genetic Engineering Prepared human gene Next. others will take up no plasmds at all or ones without antibiotic resistance genes. We need to use a µshotgun¶ approach and incubate thousands of plasmids with hundreds of bacterial cells Some cells will take up the recombinant plasmid. the human gene will fit into the cut in the plasmid so that the green µT¶ gene will no longer work correctly. Genetic Engineering Agar containing penicillin Colonies growing from single cells that are resistant to penicillin Genetic Engineering An agar plate containing Penicillin is used to allow only those cells which have taken up a suitable plasmid to survive and divide. these colonies are sub-cultured onto agar containing tetracycline. We are interested in those cells which WON¶T grow in the presence of Tetracycline 2 . we can make a cut in the midst of ONE of these antibiotic resistance genes. If all goes according to plan.

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.2/14/2012 Genetic Engineering These cells must have intact T genes Genetic Engineering These cells must have intact P genes and defective T genes Next. This colony will probably have the correct plasmid to produce the product from the human gene. these colonies are sub-cultured onto agar containing tetracycline. eg growth hormone 3 .

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