Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University

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

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

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

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

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 theGenetic engineer. Aulani "GE" Presentation 1

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) Aulani "GE" Presentation 1

7. Genetic Engineering P Cut here T By using special enzymes, we can make a cut in the midst of ONE of theseantibiotic resistance genes.In this example, we will cut open the ‘T’ gene Aulani "GE" Presentation 1

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 plasmidso that the green ‘T’ gene will no longer work correctly. Aulani "GE" Presentation 1

9. Intact P gene and ‘defective’ T gene No P or T gene P and T Genes intact Genetic Engineering As plasmids are extremely small, we cannot tell by looking which ones have gotthe human gene in the right place. We need to use a ‘shotgun’ approach andincubate thousands of plasmids with hundreds of bacterial cells Aulani "GE" Presentation 1

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 antibioticresistance genes. Aulani "GE" Presentation 1

11. Colonies growing from single cells that are resistant to penicillin Genetic Engineering Agar containingpenicillin An agar plate containing Penicillin is used to allow only those cells which havetaken up a suitable plasmid to survive and divide. These cells must have resistanceto Penicillin Aulani "GE" Presentation 1

12. 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 Aulani "GE" Presentation 1

13. 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. Aulani "GE" Presentation 1

14. 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 Aulani "GE" Presentation 1

15. to be continued... Aulani "GE" Presentation 1