Genetic Engineering Lab

1. Genetic Engineering Lab Bio 101A April 10, 2008

2. Describe your results from the PCR lab. Was your sample GMO? How do you know? • Describe differences between prokaryotes and eukaryotes.

3. Brief Overview of Lab Objectives • Obtain Bacterial DNA (plasmids-pAMP and pKAN) • Cut DNA into specific pieces using special enzymes (restriction enzymes- BamHI; HindIII) • Measure size of pieces cut by enzymes (gel electrophoresis) • Glue pieces together using other enzymes (DNA ligase) • Take glued pieces and put them into another bacterium (plasmid transformation of E. coli) • Separate bacteria with plasmid from those without (antibiotic selection)

4. Today’s Objectives • Obtain Bacterial DNA (plasmids-pAMP and pKAN) • Cut DNA into specific pieces using special enzymes (restriction enzymes- BamHI; HindIII)

5. Schedule • 9am- 910: Book check • 910-915: Review questions • 915-935: Introduction to lab • 935-10am: Set up restriction digest/cleanup • 10am-11am: restriction digest • 10am-11am: Chi square discussion/practice • 11am- refrigerate samples

6. Lab Concepts in Detail

7. Two Types of DNA in E. coli Chromosomal DNA – necessary for cell survival; circular, double-stranded Plasmid DNA – extrachromosomal DNA (“bonus material”) useful for experimental manipulation; circular, double-stranded

8. Plasmids contain nonessential (but important) genes

9. β-lactamase can destroy penicillin and other β-lactam antibiotics

10. Kanamycin interferes with Ribosomes • 30S ribosomal subunit is affected • Causes frameshift in translation • Toxic to humans

11. C G G A T C C A G C C T A G G T Plasmids can be cut with restriction enzymes Enzymes homodimerize to make symmetrical cuts BamHI GATCCA GT CG GCCTAG “sticky ends”

12. Restriction Enzymes cut very specific sequences of DNA

13. Cell containing gene of interest Bacterium Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Plasmid DNA manipula-tion is at the heart of biotech-nology Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

14. λ Phage is a temperate bacteriophage • Infects E. coli • Genome is 46,000bp long • dsDNA • Sequence is known • HindIII-digested genome is used as a molecular marker (ladder)

15. λ Phage digest is a common marker • HindIII digest of phage genome always yields the same bands

16. Draw pictures of what you expect in the microfuge tubes from last week. Include as much detail as possible. What did the plasmids look like? What do they look like now? What else is in the tube?

17. Objective(s) of the lab • 1. Digest pAMP and pKAN with BamHI and HindIII restriction enzymes • 2. Determine size of plasmids using electrophoresis • 3. Create double antibiotic resistant plasmid using DNA ligase • 4. Transform E. coli with new plasmid • 5. Select for transformants using antibiotic media plates

18. Today’s Objectives • Ligate pAMP fragment to pKAN fragment • Determine fragment sizes using electrophoresis with HindIII λ phage digest

19. Schedule • 8:10-8:20 Lecture spiel • 8:20-8:55 Denature/Pour gel • 9-10:15 Set up/Run gel • 9:30-10:15 Discuss last quiz/Drosophila/Chi-square • 10:15-10:30 Visualize gel • 10:30-10:50 Create semilog graphs of digest/determine fragment sizes • 10:50-11 clean up

20. BamHI BamHI kanR pKAN pAMP HindIII ampR HindIII Ori Ori Restriction digest Ori BamHI Ori ampR BamHI HindIII HindIII kanR BamHI HindIII Ligation ) BamHI kanR HindIII ampR Ori

21. T4 is a Lytic bacteriophage • Why might a lytic bacteriophage need DNA ligase?

22. Undigested plasmids are often supercoiled • Supercoiling- increased or decreased number of twists/bp • Can be caused by topoisomerases (type I and type II)

23. Topoisomerases can cut DNA once or twice • Either way can increase or decrease supercoiling • Dimers can be made or removed by topoisomerases

24. Supercoiled, relaxed and linear DNA do not run equally • Why is supercoiled faster than linear? • Why are dimers slower than monomers?

25. Week 3: Transforming Bacteria Review Questions • What is our objective for the lab? • What was accomplished for this task last week? • How did what was done last week further our objectives for the lab? • Define the following: • Plasmid • Ligase • Restriction Enzyme

26. Week 3: Transforming bacteria Outline for today • 15 min. introduction • Transformation protocol (45 min.) • Incubation (60 min) • During incubation- Outline of selection (20 min); completion of worksheet (due Wed. noon) • Plating- 30 min. • Predict plating results- (10 min)

27. Bacterial Transformation • We will use chemically competent E. coli cells • CaCl2, ice incubation, and heat shock facilitate the process

28. Procedure • Add 200 uL of competent bacteria to +LIG vial • Add 200 uL bacteria to any controls • Gently mix • Incubate on ice for 20 min

29. Procedure, cont. • Heat shock for 90 sec. • Place back in ice for min. 60 sec. • Add 800uL sterile LB to tube • Incubate on shaker for 60 min.

30. Sterile technique reminder • Bacteria are ubiquitous • Flame kills bacteria • Any contaminants will compete with our bacteria of interest

31. Micropipettors Review Are fragile Expensive Precise They depend on correct usage for accuracy

32. Competent cells • Transformation rate in normal cells is low • Transformation rate in competent cells is higher • We use CaCl2 to make cells chemically competent

33. How can we determine if our transformation was a success?

34. Selecting for transformants • Antibiotic-infused agar media permits only resistant bacteria to grow • Our plasmids confer specific antibiotic resistance