1. Agenda – Applying DNA knowledge to diabetes
Warm-up:
Attractive & professional thank-you notes
Diabetes, Insulin, and rDNA
Review
Recombinant DNA
Plasmids
Recombinant Paper Plasmid

2. Recombinant DNA project
Notes on what you have learned – due tomorrow
Electronic presentations
Engineering and Biotechnology by Dong Min Kim on Prezi

3. Diabetes and Biotechnology
Biotechnology solutions
Recombinant DNA

4. Questions- What is diabetes?
Why is there growing concern about diabetes?
What is the role of insulin?
What is the role of biotechnology in the treatment of diabetes?

5. Biotechnology & Diabetes Treatment
Read Calorie-Coated Diabetes

6. Beta Cell in Pancreas

7. Pancreas, thymus

8. Cell Biology
Of Insulin
Response

9. Cell Biology
Of Insulin
Response

10. Cell Biology Of Insulin Response

11. Insulin Gene is on the 11th Chromosome (short arm)
It contains 153 bases.
A small, simple protein

12. Recombinant DNA with insulin gene inserted
Recombinant DNA: A desired gene to the plasmid. Bacteria is transformed to accept the plasmid.

13. Chromosome & Plasmid

14. Interesting facts about plasmids
What do plasmids do in bacteria?
Why are plasmids used in recombinant DNA?
In nature?

15. Interesting facts about plasmids
What do plasmids do in bacteria?
Why are plasmids used in recombinant DNA?
Extra genes
Not essential for living
Benefit for survival under certain conditions
In nature, some plasmids
Antibiotic genes
Antibiotic resistant genes
Bacteria can be transformed by adding circular DNA but not linear
Exonuclease:
Breaks down genes in a linear process
Outside to the inside

16. Making a Recombinant DNA Product
What are the steps needed?
Paper plasmids -
Construction of the pAMP and pKAN Plasmids
Questions

17. Genetic Engineering –Recombinant DNA How?
Identify a molecule produced by a living organism
Isolate the instructions (DNA sequence = gene)
Put the instructions into another cell or organism
Allow the cell to replicate
Harvest the desired product

18. The Vector (E. coli bacteria).

19. What is needed to produce a product with recombinant DNA?

20. Recombinant DNA Insulin
Identify a insulin gene in humans
Use restriction enzymes to isolate the gene in a DNA fragment
DNA fragment is added to another DNA source = vector
such as plasmids of bacteria or yeast
Recombinant DNA is placed in a host cell
As the host cell divides (replicates), the rDNA also replicates
Harvest, purify, test & market

21. What we need to learn about:
Bacteria (Vector)
Copying cells - Mitosis
Proteins
How are enough insulin is made? PCR

22. Recombinant DNA & Cloning

23. What is DNA cloning?
When DNA is extracted from an organism, all its genes are obtained
In gene (DNA) cloning a particular gene is copied (cloned)

24. Why Clone DNA? e.g. gene defects related to specific diseases
A particular gene can be isolated and its nucleotide sequence determined
Control sequences of DNA can be identified & analyzed
Protein/enzyme/RNA function can be investigated
Mutations can be identified
e.g. gene defects related to specific diseases
Organisms can be ‘engineered’ for specific purposes
e.g. insulin production, insect resistance and more

25. How is DNA cloned?, I DNA is extracted- here from blood
Blood sample
DNA is extracted- here from blood
Restriction enzymes, e.g. EcoR I, Hind III, etc., cut the DNA into small pieces
Different DNA pieces cut with the same enzyme can join, or recombine.
DNA
Restriction enzymes

26. DNA Cloning, II
Bacterial plasmids (small circular DNA additional to a bacteria’s regular DNA) are cut with the same restriction enzyme
A chunk of DNA can thus be inserted into the plasmid DNA to form a “recombinant” molecule

27. DNA cloning, III
The recombinant plasmids are then mixed with bacteria which have been treated to make them “competent”, or capable of taking in the plasmids
This insertion is called transformation

28. DNA Cloning, IV
The plasmids have naturally occurring (or inserted) genes for antibiotic resistance
Bacteria containing plasmids with these genes will grow on a medium containing the antibiotic- the others die, so only transformed bacteria survive

29. DNA Cloning, V
The transformed bacterial cells form colonies on the medium
Each cell in a given colony has the same plasmid (& the same DNA)
Cells in different colonies have different plasmids (& different DNA fragments)

30. Screening, I Screening can involve:
Phenotypic screening- the protein encoded by the gene changes the color of the colony
Using antibodies that recognize the protein produced by a particular gene

31. Screening, II
3. Detecting the DNA sequence of a cloned gene with a probe (DNA hybridization)

32. rDNA project Choice of format: Presentation: Equivalent to Test Grade
PowerPoint Presentation with notes
Globster (electronic poster)
Museum boxes
Presentation:
Process to make rDNA
Process before rDNA
Benefits
Disadvantages or bioethical issues
Regulation
Equivalent to Test Grade

33. Additional Resources
Discovery and Applications of REs -