1. Genetic Engineering First, the nucleus of human cells are burst

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

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

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

5. 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 the Genetic engineer.

6. 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)

7. Genetic Engineering P T Cut here
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

8. 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.

9. Genetic Engineering 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

10. 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.

11. 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

12. 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

13. 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.

14. 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