Genetic Engineering

1. Genetic Engineering

2. A. Selective Breeding Choosing the best of the best (characteristics) & breeding the 2 organisms to achieve the desired results -- perfection I. Selective Breeding vs. Genetic Engineering

3. Direct manipulation of genes to achieve desired goal The process is called Recombinant DNATechnology B. Genetic Engineering Because we can cut DNA where we want, we can mix DNA of different organisms. Organisms are called transgenic.

4. Example: Creation of Insulin for diabetics “Old days” Problem: Some people were allergic to pig products. Caused bad side effects.

5. Various genes from plants that are desirable & enhance profit have been used: Traits include: Drought resistant Pest resistant Tolerance to cold Yield Great Taste II. Genetic Engineering in Crops A. Genetically Modified Foods (GMF)

6. B. Genetic Engineering is a faster more reliable method for increasing the frequency of a specific allele in a population.

7. Plasmid A small circular piece of DNA, common in bacteria, that genetic engineers use as a vector. III. Bacterial Transformation A. Terminology

8. “Competent” bacterial cells Vector: When bacteria cells are put through a process that allows their membranes to “take-up” DNA (for example, a plasmid). A carrier or transport. We will use a plasmid as the vector of a couple of foreign genes into a bacteria.

9. Restriction Enzymes Enzymes that cut DNA at SPECIFIC sequences. Recombinant DNA When a gene, or segment of DNA, from one organism is inserted into the DNA of another organism. The new cell is said to be “transformed”. The DNA is called recombinant.

10. DNA “sticky-ends” After a restriction enzyme cuts DNA, “sticky ends are a stretch of unpaired nucleotides at the cut.

11. Bacteria Prokaryote, one-celled organism. E-coli is the bacteria that will be used in our lab. Found in our intestines. Helps us breakdown food. It likes the body’s temperature of 37 C. Used extensively in recombinant DNA research.

12. “Luria broth or agar” A nutritionally rich medium that is primarily used for the growth of bacteria.

13. Immediately put in hot water bath “Heat-shock” First put bacteria on ice Helps the bacteria to “suck-in” the plasmid (with it’s recombinant DNA). An antibiotic Anti = against; bio = life. Antibiotics kill bacteria. We will use Ampicillin.

14. “Lawn” of bacteria”? A continuous cover of bacteria on the surface of a growth medium

15. What is a “colony” of bacteria? Separate masses of bacteria (clones)

16. A fluorescent gene • from a jellyfish. • 2. A gene that will make • the bacteria resistant • to ampicillan. The 2 genes that will be inserted on the Plasmid (which will be inserted into the E-Coli:

18. Jones Block 1 LB/AMP + LB/AMP + • Luria Broth mixed with Ampicillin • (+) Bacteria with plasmid will be • spread on this plate

19. Jones Block 1 LB/AMP - LB/AMP - • Luria Broth mixed with Ampicillin • Bacteria with no plasmid will be • spread on this plate.

20. Jones Block 1 LB - LB - • Luria Broth with no Ampicillin • Bacteria with no plasmid

21. Luria Broth Only No Ampicillin Bacteria with no plasmid (-) LB Growth or No Growth? Growth Why? “Food” available. No antibiotic to kill it. Fluorescent? Ampicillin Resistant? NO. No plasmid with those genes.

22. LB/AMP Luria Broth with Ampicillin Bacteria with No Plasmid Growth or No Growth? NO Growth Why? No plasmid with antibiotic resistant gene

23. LB/AMP Luria Broth with Ampicillin Bacteria with Plasmid Growth or No Growth? Growth. Why? Bacteria has plasmid with antibiotic resistant gene Fluorescent? Yes.

24. Scorpion- Natural Light Scorpion- UV Light In the Dark Bioluminescence vs. Fluorescence Bioluminescence Fluorescence Natural Light A fluorescent organism absorbs light at one wavelength (UV) and a re-emits the light at a visible wavelength = color Bioluminescent organism produces its own light.

25. Sea Pansy Copepods small planktonic crustacean Jellyfish (Aequorea victoria) Cerianthus Tube Anemone Many Organisms Can Fluoresce

26. OSAMU SHIMOMURA Co-winner of Nobel Prize Green Fluorescent Protein Red Fluorescent Protein They all contain fluorescent proteins

27. The FP gene is inserted right after the gene for the protein, before the stop codon. The protein of interest AND the FP are translated together. Now, the scientist can visualize the protein of interest (its location) and measure the amount of protein translated (how much it fluoresces). http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP4.htm

28. Why would a researcher use a fluorescent protein? Think of GFP as the microscope of the twenty-first century. Using GFP we can see when proteins are made, and where they can go. This is done by joining the GFP gene to the gene to the protein of interest so that when the protein is made it will have GFP hanging off it. Since GFP fluoresces, one can shine light at the cell and wait for the distinctive green fluorescence associated with GFP to appear.

29. GFP in Sperm Helps determine which sperm cells “mate” with egg In most species of insects, birds, and some arachnids, the second male to copulate with a female, fathers most of her offspring. How does the sperm of the last male to mate maximize its chances of being fertilized? By tagging the sperm of the first mate with GFP, researchers were able to distinguish between sperm released by the first and second partners. They found that the first male's sperm is displaced by that of the second partner.

30. Transgenic Tobacoo Plant

31. Transgenic Zebra Fish

32. Under normal light Under Black Light

33. Transgenic Mice

34. Transgenic Pork This gives a whole new meaning to green eggs & ham!

35. Transgenic Fish Embryos Normal Light UV Light

36. GFP-tubulin GFP-chromatin GFP-membrane pro pro met met met ana ana ana telo telo telo cyto Visualizing FPs in Live Worms ……analyzing mitosis from 3 perspectives:

37. Optical Illusion If you stare at this picture for 30 seconds you will start to see details of a giraffe.