1. Aulani "GE" Presentation 1 Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University Genetic Engineering

2. Aulani "GE" Presentation 1 First, the nucleus of human cells are burst Human cell Nucleus

3. Aulani "GE" Presentation 1 The chromosomes are cut up into small fragments and the required gene identified. Chromosome fragments Fragment containing required gene

4. Aulani "GE" Presentation 1 Cytoplasm Bacterial chromosome Bacterial cell wall Plasmid Structure of a typical bacterium

5. Aulani "GE" Presentation 1 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. Aulani "GE" Presentation 1 Genetic Engineering In the above plasmid, the YELLOW gene is one that gives the bacterium resistance to one antibiotic (eg Penicillin). P T The GREEN gene gives resistance to a different antibiotic (eg Tetracycline)

7. Aulani "GE" Presentation 1 Genetic Engineering 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 P T Cut here

8. Aulani "GE" Presentation 1 Genetic Engineering 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. Prepared human gene

9. Aulani "GE" Presentation 1 Genetic Engineering 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 No P or T gene Intact P gene and ‘defective’ T gene P and T Genes intact

10. Aulani "GE" Presentation 1 Genetic Engineering 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. Required cell Cell with P and T intact Cell with neither P or T

11. Aulani "GE" Presentation 1 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 cells must have resistance to Penicillin Agar containing penicillin Colonies growing from single cells that are resistant to penicillin

12. Aulani "GE" Presentation 1 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. Aulani "GE" Presentation 1 Next, these colonies are sub-cultured onto agar containing tetracycline. These cells must have intact T genes These cells must have intact P genes and defective T genes

14. Aulani "GE" Presentation 1 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

15. Aulani "GE" Presentation 1