Genetic Engineering First the nucleus of human cells

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

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

Genetic Engineering Cytoplasm Bacterial cell wall Plasmid Bacterial chromosome Structure of a typical bacterium

Genetic Engineering 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.

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)

Genetic Engineering 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

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 plasmid so that the green ‘T’ gene will no longer work correctly.

Genetic Engineering 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

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 antibiotic resistance genes.

Genetic Engineering 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

Genetic Engineering 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

Genetic Engineering 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.

Genetic Engineering 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