1. Exam 2 T 10/30 at 7:30-9pm Review Th 10/25 at 5-7pm in WRW 102 and/or in class Bonus #1 due 10/25 in class

2. Genetic Engineering: Direct manipulation of DNA

3. Bacteria can be modified or serve as intermediates

4. a typical bacteria Bacterial DNA plasmid DNA

5. A typical bacterial plasmid used for genetic engineering

6. Moving a gene into bacteria via a plasmid

7. Bacterial DNA plasmid DNA What problems exist for expressing eukaryotic gene in bacteria?

8. Reverse transcriptase can be used to obtain coding regions without introns.

9. Fig 20.14 After RT, PCR will amplify the gene or DNA

10. Moving a gene into bacteria via a plasmid RT and PCR

11. Restriction Enzymes cut DNA at specific sequences

12. Restriction enzymes cut DNA at a specific sequence

13. Fig 20.2 Cutting the plasmid and insert with the same restriction enzyme makes matching sticky ends

14. A typical bacterial plasmid used for genetic engineering

15. Using sticky ends to add DNA to a bacterial plasmid Fig 20.2

16. If the same restriction enzyme is used for both sides, the plasmid is likely to religate to itself.

17. Fig 20.2 The plasmid is treated with phosphatase to remove the 5’-P, preventing self- ligation

18. Fig 20.8 Transformation of bacteria can happen via several different methods.

19. Fig 7.2 Bacteria can take up DNA from the environment

20. Fig 20.8 Transformation of bacteria can happen via several different methods all involving perturbing the bacterial membrane: Electroporation Heat shock Osmotic Stress

21. How can you know which bacteria have been transformed, and whether they have the insert?

22. Figure 20-5 Fig 20.5 Resistance genes allow bacteria with the plasmid to be selected. Bacteria with the resistance gene will survive when grown in the presence of antibiotic

23. Figure 20-5 Fig 20.5 Is the insert present? Plasmids with the MCS in the lacZ gene can be used for blue/white screening…

24. A typical bacterial plasmid used for genetic engineering

25. Figure 20-5 Fig 20.5 Intact lacZ makes a blue color when expressed and provided X-galactose

26. Figure 20-5 Fig 20.5 When the lacZ gene is disrupted, the bacteria appear white

27. Blue/white screening: Transformed bacteria plated on antibiotic and X- gal plates. Each colony represents millions of clones of one transformed cell.

28. Successful transformation will grow a colony of genetically modified bacteria Fig 20.4

29. Inserting a gene into a bacterial plasmid RT and/or PCR

30. Millions of Hectares Texas = 70 ha Bacteria can be used to transform plants Global area planted with GM crops http://www.gmo-compass.org/eng/agri_biotechnology/gmo_planting/257.global_gm_planting_2006.html

31. Agrobacterium infect plants, inserting their plasmid DNA into the plants genome.

32. Fig 20.24 Agrobacterium infect plants, inserting their plasmid DNA into the plants genome.

33. Fig 20.25 By replacing the gall forming genes with other DNA when the Agrobacterium infect a plant, it will insert that DNA into the plant.

34. Fig 20.26 The generation of a transgenic plant Grown on herbicide

35. How do you know whether the gene you want to express has the correct sequence?

36. DNA sequencing

37. The structure of 2 ’,3 ’ -dideoxynucleotides Fig 20.15

38. Fig 20.16 The dideoxy sequencing method

39. Fig 20.16 The dideoxy sequencing method

40. Fig 20.16 Gel produced by the dideoxy sequencing method

41. Fig 20.17 Computerized sequencers use a similar method

42. Creation of Caenorhabditis elegans transgenes Figure 20-28

43. Creation of Drosophila melanogaster transgenes using a transposon Fig 20.29

44. Creation of Mus musculus transgenes Figure 20-30 Fig 20.30

45. …now enjoy making Frakencritters.

46. Exam 2 T 10/30 at 7:30-9pm Review Th 10/25 at 5-7pm in WRW 102 and/or in class Bonus #1 due 10/25 in class