1. BIOTECHNOLOGY -intentional manipulation of genetic material of an organism

2. determines the characteristics of all living organisms. occurs in most cells of all organisms composed of four different nucleotides in different combinations each cell in the human body contains more than 3 BILLION letters Deoxyribonucleic Acid (DNA)

3. Four bases: Adenine Thymine Guanine Cytosine 2 bonds 3 bonds Sugar and phosphate backbone Double helix structure (two spirals around each other)

4. The only difference between living organisms is the amount and order of the four nucleotide bases.

5. Genome: the entire sequence of DNA Gene: the part of the code that corresponds to a protein *genes can be transferred from one organism to another*

7. BIOTECHNOLOGY The intentional manipulation of genetic material of an organism Why would we want to do this? To study cellular processes of an organism –E.g. Glowing gene from jellyfish to tobacco plant To give one organism the trait(s) of another –E.g. Anti-freeze from fish blood into strawberries to survive through early frosts

9. Part 1: Manipulating Bacteria: The Making of a Plasmid

10. Plasmid: - a small circular piece of extra-chromosomal bacterial DNA, able to replicate - bacteria exchange these plasmids to share DNA - E.g. antibiotic resistance genes

11. Since plasmid is made of DNA it can code for genes, ex. antibiotic resistance, and can carry specific sequences of DNA Specific DNA sequences can be recognized by enzymes called restriction endonucleases

12. Restriction Endonucleases/Restriction Enzymes enzymes that are able to cut double- stranded DNA into fragments at specific recognition sites in DNA sequences Ex. EcoRI: 5’-GAATTC-3’ 3’-CTTAAG-5’

14. Restriction enzymes can create “sticky ends or “blunt ends”

15. Sticky Ends fragment end of a DNA molecule with a short single-stranded overhang Blunt Ends fragment end of a DNA molecule with no overhang Once made, the ends can be re-joined together by other enzymes ("enzyme glue")

16. To Make a Recombinant Plasmid: 1.Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends. Insert 2.Combine the sticky ends using ligase. ligase: enzyme used to join DNA together 3. Introduce the recombinant plasmid into bacteria.

17. Making a Recombinant Plasmid

19. Bacterial Transformation - - - - - - - - + + + + + - ++ -++ - - ++ + + + + + +-++-+ phospholipid bilayer plasmid Ca 2+ ions introduction of foreign DNA into a bacterial cell plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell

20. Following transformation bacteria are grown in medium with antibiotic… Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

21. Example plasmid: Origin of Replication: the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate where the plasmid starts to duplicate itself

22. Part 2: Where do we get our insert sequence? From someone else’s DNA –ex. fish gene in strawberries, –jellyfish gene in plants Make it!

23. In order to do these things, we need a way to make many copies of the genes we want

24. easy to grow no ethical issues small genome easy to manipulate Using Bacteria as Production Factories

26. Making an insert: Polymerase Chain Reaction

28. Common uses of biotechnology: 1.Making "stuff” proteins, enzymes, medication, etc. can be produced by engineered bacteria! Food can be altered to have new traits Cloning (therapeutic and reproductive) 2.Genetic screening crime cases, relationship, genetic screening, etc. 3. Gene Therapy

29. Therapeutic cloning used to produce tissue that is identical to the donor, to prevent rejection

30. Reproductive Cloning creates an organism with the same genetic material (DNA) as the original organism – an EXACT COPY of the donor

32. Dolly the Sheep the first cloned sheep

33. Ex. RFLP: Restriction Fragment Length Polymorphism Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

34. Gene therapy - desired gene is inserted into cell's nucleus using a retrovirus as a carrier - defective gene replaced by functional gene

35. Ex. ADA deficiency -adenosine deaminase deficiency -little immunity with low chances of recovery - the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system