1. Exam 2 M 10/29 at 7-8:30pm in UTC 2.102A
Review Th 10/25 at 5-7pm in WRW 102
Bonus #1 due now
Last day to drop with a ‘Q’…10/24

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

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

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

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

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.
Fig 20.2

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

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

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

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

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

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

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

24. A typical bacterial plasmid used for genetic engineering

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

26. Figure 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
Inserting a gene into a bacterial plasmid