1. MOLECULAR BIOLOGY IN ACTION In this project, students will use what they have learned in the previous courses to complete a larger multi-step molecular biology experiment. They will have the chance to assemble a recombinant plasmid, clone this DNA, and analyze products through restriction analysis

2. Lab 6.1.2 1. Module I : The ligation of a plasmid vector with a fragment containing the kanamycin resistance gene. 2. Module II: Introduction of the recombinant DNA into E. coli cells by transformation and selection of transformants 3. Module III: Picking and growth of Kanr transformants 4. Module IV: Extraction of supercoiled recombinant plasmid DNA 5. Module V: Restriction enzyme analysis

3. Module 1: ligation of a plasmid vector Insert a kanamycin resistance gene (1300bp) into the 3000bp plasmid vector that already contains an ampicillin resistance gene BOTH already cut with EcoRi

4. The plasmid vector Is a 3000 base pair plasmid. Possesses a single restriction site for the enzyme EcoRI. Contains an ampicillin resistance gene. Has previously been cut with EcoRI to open the circle and create a linear piece with available sticky ends. The kan r Gene The cloned fragment encoding the gene is approximately 1300 base pairs in length and possesses EcoRI generated ends. 813 base pairs code for the polypeptide. The code for the polypeptide is flanked by a 144bp promoter and a 344bp ribosomal binding sequence

5. Vector + Insert

6. E= EcoRI, P = PvuII, and C = ClaI

8. The plasmid can undergo reclosure without an insert The insert can adhere in two orientations (50/50) More than one insert can enter the circle (and each of these can be in a different orientation) The full length vector can link to other copies of the vector in a linear chain referred to as a concatamer. Some of these concatamers may actually contain the kanamycin insert as well – they have just never reformed a circular shape

9. Module 1: ligation of a plasmid vector 1A: LIGATION (Overview at left) 1B: AGAROSE GEL 1C: STAINING & VIEWING

10. Module 1A: Ligation prep (30m)

11. MODULE 1A: LIGATION OF A PLASMID VECTOR TO THE KANR GENE FRAGMENT

13. Verification of Ligation Success of ligation is verified by gel electrophoresis. The vector + insert should be a 4300bp plasmid. The conformation of the plasmid DNA determines distance travelled. Complex banding patterns demonstrate the presence of various ligation products. Transformation acts as a purification step and separates this complex mixture.

14. Module 1B prep: Electrophoresis (10m)

15. MODULE 1B: AGAROSE GEL ELECTROPHORESIS

16. MODULE 1B: AGAROSE GEL ELECTROPHORESIS, CONT

17. Module 1C prep: Staining (30m)

18. MODULE 1C: STAINING

19. Module 1: Results & Analysis

21. The ligation reaction will be used in module II. The T4 DNA Ligase Tube can be stored at -20C

22. Module II: Introduction of the Recombinant DNA into E.coli Cells The new plasmid is transferred to E.coli cells through the process of bacterial transformation. Cells are plated on media containing kanamycin. Cells must contain the new plasmid (containing the kan r gene) to grow on this media. This process identifies cells with the recombinant plasmid.

23. Module 2: transformation of recombinant DNA into e.coli 1A: LIGATION (Overview at left) 1B: AGAROSE GEL 1C: STAINING & VIEWING

24. Module 2: Prep

25. Module 2 prep: E. coli source plates

28. After Step 14 the samples can be placed in the refrigerator and then centrifuged and plated the next class period. This may help alleviate some of the stress of a shorter lab period.

30. Module III: Picking and Growth of kan r Transformants A single transformed colony is picked, transferred to liquid media containing kanamycin, and allowed to incubate. The bacteria reproduce, making new copies of the plasmid in each cell and cloning the DNA of interest.

31. Module IV: Extraction of Supercoiled Recombinant Plasmid DNA The plasmid must be removed from the cell to be examined. SDS, a detergent, disrupts cell membranes and denatures proteins. RNAse, an enzyme that breaks down RNA, helps remove residual RNA. While linear and nicked plasmid DNA can undergo strand separation as they are released from solution, supercoiled plasmid DNA remains intact.

32. Module V: Restriction Enzyme Analysis Digestion of the plasmid with restriction enzymes identifies features of the ligated plasmid. Features of the plasmid map suggest digestion results for the possible recombinants. Expected digestion results are compared to gel electrophoresis banding patterns.

33. Modules 3-5: Overview 3: Culturing of KanR Transformants 4: Extraction of Recombinant Plasmid DNA 5: Restriction Enzymes & Analysis via Electrophoresis

34. Module 3: Prep

35. Module 4: Prep

36. Module 5: Prep

45. Results of Ligation This ligation can prompt several possible outcomes. The vector may close with no insert. The vector may close with one insert (in one of two orientations). The vector may close with multiple inserts (each insert in one of two orientations). The plasmid contains the following features to be used as reference: EcoRI sites on each side of the new fragment A single PvuII site on the plasmid A single ClaI site on the insert Images provided courtesy of Edvotek® - www.edvotek.com

46. Analyzing Results Refer to your plasmid drawings in Conclusion question 2 of Project 6.1.2. Predict what you might see in each of the following lanes for each plasmid you have drawn. Lane 1: Standard DNA fragments Lane 2: Plasmid Vector (nonrecombinant) Lane 3: Recombinant Plasmid Vector (uncut) Lane 4: Recombinant Plasmid Vector (cut with EcoRI) Lane 5: Recombinant Plasmid Vector (cut with PvuII) Lane 6: Recombinant Plasmid Vector (cut with PvuII & ClaI)

47. Result Option 1

48. Possible Result: Single Insert – 5’ to 3’ Orientation

49. Lane 6 helps identify the orientation of the fragment. The ClaI site and the PvuII site are a good distance apart (almost the entire length of the 1300bp fragment). Two bands are visible that correspond to a little less than 1300bp and over 3000bp. Images provided courtesy of Edvotek® - www.edvotek.com

50. Result Option 2

51. Possible Result: Single Insert – 3’ to 5’ Orientation Image provided courtesy of Edvotek® - www.edvotek.com

52. Possible Result: Single Insert – 3’ to 5’ Orientation Lane 6 helps identify the orientation of the fragment. The ClaI site and the PvuII site are now much closer together. Thus, two bands are visible that correspond to the small fragment (approximately 450 bp) and the remaining plasmid piece (approximately 3800 bp). Images provided courtesy of Edvotek® - www.edvotek.com

53. Result Option 3

54. Possible Result: Double Insert – 5’ to 3’ Orientation Image provided courtesy of Edvotek® - www.edvotek.com

55. Possible Result: Double Insert – 5’ to 3’ Orientation Plasmid vector cut with PvuII helps identify the number of fragments inside the vector. Lane 5 reveals one band corresponding to the open circle of DNA at approximately 5600bp. The original plasmid was 3000bp. Each fragment is 1300bp. Thus, this plasmid must have incorporated two fragments. What other enzyme could help identify number of fragments? Images provided courtesy of Edvotek® - www.edvotek.com

56. Possible Result: Double Insert – 5’ to 3’ Orientation Plasmid vector cut with PvuII and ClaI helps identify the orientation of the fragments. Can you explain how?

57. Possible Result: Vector without Insert + Recombinant Plasmid with 3’ to 5’ Single Insert

58. It is possible for the bacteria to carry more than one form of the plasmid. On its own a cell containing a plasmid without an insert would not have grown on kanamycin media. However, since it is housed in a cell that also contains a plasmid with the resistance gene, it is visible.

59. Possible Result: Vector without Insert + Recombinant Plasmid with 3’ to 5’ Single Insert Lane 5 provides an indicator that two different plasmids exist in the sample. Digestion with PvuII simply opens the plasmid ring. In samples with one plasmid, one linear fragment results. In this gel Lane 5 shows two bands, consistent with the size of the plasmid DNA from the original plasmid (3000bp) and a plasmid with the insert (4300bp). The orientation of the recombinant plasmid can still be verified with the double digest in Lane 6. How? Images provided courtesy of Edvotek® - www.edvotek.com