WSSP-14 Chapter 1 Vectors and Libraries

1. WSSP-14 Chapter 1 Vectors and Libraries

2. Today you will start doing something in the lab called "Molecular Cloning" Or "Genetic Engineering" Or "Recombinant DNA Technology" These techniques will allow you to study and manipulate individual genes

3. For many years, biochemists had tried to purify genes. But they were frustrated because they are hard to purify.

4. Because genes are composed of A’s, C’s, G’s, and T’s, they all pretty much are chemically alike. Also genes are parts of chromosomes. Chromosomes break easily and randomly, often in the middle of genes. So how did scientists eventually purify individual genes?

5. Genetic Engineering Nobel Prizes Paul Berg Herb Boyer (Genetech) Stanley Cohen

6. Vector containing insert is transformed into E. coli for amplification and purification Amplify and Prep p. 2-1

7. Vectors In order to study a DNA fragment (e.g., a gene), it needs to be amplified and eventually purified. These tasks are accomplished by cloning the DNA into a vector. A vector is generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli (can be a virus). p. 1-1

8. Plasmids Circular DNA molecules found in bacteria Replicated by the host’s machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication. Some plasmids are present in E. coli at 200-500 copies/cell p. 1-4

9. Plasmids also contain selectable markers. Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. Plasmid Engineering p. 1-4

10. Transforming plasmids into bacteria Very inefficient: less than 1/1000 cells are transformed with the circular plasmid (linear does not transform) p. 1-2

11. Transforming plasmids into bacteria Need to treat cells with Ca++ to transform plasmid Normal Cell (negatively charged membrane) Plasmid Ca++ Cell treated with CaCl2 (more postively charged membrane)

12. Transforming plasmids into bacteria Very inefficient: less than 1/1000 cells are transformed with the plasmid How do you identify the few cells with the plasmid?

13. Plasmid Engineering • Plasmids also contain selectable markers. • Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. • Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). • Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. p. 2-2

14. Plate cells on media with antibiotic Kills cells without the plasmid

15. Cloning a DNA fragment Dead Cells Colony p. 1-2

16. Safety Features Modern cloning plasmids have been engineered so that they are incapable of transfer between bacterial cells Provide a level of biological containment. Naturally occurring plasmids with their associated drug resistance genes are responsible for the recent rise in antibiotic-resistant bacteria plaguing modern medicine. p. 1-3

17. LacZb-galactosidase, Jacob & Monod X-gal

18. Screening for Inserts p. 1-3

19. Transform plasmid into bacteria

20. DNA Libraries • DNA library - a random collection of DNA fragments from an organism cloned into a vector • Ideally contains at least one copy of every DNA sequence. • Easily maintained in the laboratory • Can be manipulated in various ways to facilitate the isolation of a DNA fragment of interest to a scientist. • Numerous types of libraries exist for various organisms - Genomic and cDNA. p. 1-5

21. Construction and analysis of a genomic DNA library Shotgun sequencing p. 1-5

22. Construction and analysis of a genomic DNA library

23. Construction and analysis of a genomic DNA library

24. Construction and analysis of a genomic DNA library

25. Want large clones to span the genomic DNA

26. Sequencing the human genome cost $3 billion. Efforts are being made to cut the cost of sequencing a specific human genome to $1,000 (or less)

27. We are not sequencing a genomic DNA library. We are sequencing a cDNA library What's that and what is the difference between the two?

28. We are not sequencing a genomic DNA library. We are sequencing a cDNA library What's that and what is the difference between the two? A cDNA is a copy of RNA (usually mRNA) in the form of DNA.

29. mRNA is a processed RNA transcript (in eukaryotes). It is intended to be translated into a protein.

30. Construction of a cDNA library Why are there blue colonies? p. 1-6

31. Construction of a cDNA library

32. Construction of a cDNA library ?

33. Differences between a genomic and cDNA library Genomic Library Promoters Introns Intergenic Non-expressed genes cDNA Library Expressed genes Transcription start sites Open reading frames (ORFs) Splice points p.1-7

34. Purification of mRNA Collect and grind up plants in mild denaturing solution Spin out debris (Tissue, membranes, etc) Treat with DNAse (removes DNA) Treat with Phenol (removes protein) p. 1-8

35. Synthesis of cDNA from mRNA p. 1-8

36. SfiI digestion sites of pTRiplEX2 p. 1-9

37. Cloning Duckweed cDNA fragments into the pTriplEX2 polylinker cDNA Insert p. 1-10

38. WSSP-14 Chapter 1B Plasmid Preps

39. Grow an overnight (ON) culture of the desired bacteria in 2 ml of LB medium containing the ampicillin antibiotic for plasmid selection. Incubate the cultures at 37°C with vigorous shaking. 1. Grow the bacteria amp p. 2-11

40. Naming your clones # School 01. Bayonne HS, NJ 02. Bridgewater HS, NJ 04. East Brunswick HS, NJ 05. High Point HS, NJ 06. Hillsborough HS, NJ 07. James Caldwell HS, NJ 09. JP Stevens HS, NJ 11. Montville HS, NJ 13. Pascack Hills HS, NJ 14. Pascack Valley HS, NJ 15. Rutgers Prep., NJ 16. Somerville HS, NJ 17. The Pingry School, NJ 18. Watchung Hills HS, NJ 19. West Windsor-Plains. HSS, NJ 38. Hackettstown, NJ 47. Fairlawn NH, NJ 49. Piscataway, NJ 50. The Frisch School, NJ 70. Old Bridge HS, NJ 93. The Peddie School, NJ 94. Academy of Edison, NJ 95. Acad. Of Enrichment & Adv., NJ 96. Holmdel HS, NJ 97. Robbinsville HS, NJ 98. Union City HS, NJ 103. The Hun School, NJ 104. Elmwood Park Memorital, NJ 105 North Brunswick, NJ # School 34. Science & Math Acad. MD 35. Walter Johnson, MD 59. Col. Zadok Magruder HS, MD 62. Winston Churchill, MD 81. South River HS, MD 82. Southern HS, MD 87. Kent Co HS IBALC, MD 88. Randallstown HS, MD 89. Gilman School, MD 100. Cristo Rey Jesuit HS, MD 101. Pikesville HS, MD 102. New Town HS, MD # School 65. Dougherty HS, CA 66. Modesto HS, CA 67. Tracy HS, CA 68. Waipuhu HS, HI 90. Granada High School, CA 91. Amador Valley High School, CA Your initials Year 20AV12.14 School # Clone #

41. Enter the names of the clones into your school’s Google Docs Clone Report sheet

42. The average ON contains 109 cells/ml.

43. Grown ON shaking at 37°C Grown ON on the bench at RT

44. One way to tell if your ON is fully grown is to see if you can see writing if you hold the tube up to your notes Needs to grow longer Fully Grown

45. 2a. Transfer the cells to a tube and centrifuge Transfer 1.5 ml of the culture to a microfuge tube and pellet the cells for 1 minute at full speed (12,000 rpm) in the microcentrifuge. First tap or gently vortex the glass culture tube to resuspend the cells which have settled. The culture can be transferred to the microfuge tube by pouring. (Follow steps in Lab 6) p. 1-12

46. 2a. Centrifuge the samples Balance the tubes in the centrifuge Pellet the cells for 1 minute at full speed (10,000-14,000 rpm) in the microcentrifuge.

47. 2a. Centrifuge the samples Before After Make sure there is a good size pellet

48. 2b. Remove the supernatant Remove the growth medium (supernatant) by pouring out into a waste cup. Leave the bacterial pellet as dry as possible so that additional solutions are not diluted.

49. 3a. Resuspend the cell pellet Resuspend the bacterial pellet in 200 µl of Solution I by pipeting up and down. Add 200 l of Solution I, cap the tube, and vortex on the highest setting (pipetman can be used). Look very closely for any undispersed pellet before proceeding to the next step. It is essential that the pellet be completely dispersed. Solution I contains three essential components: glucose, Tris and EDTA. Glucose and Tris are used to buffer the pH of the cell suspension. EDTA is a chemical that chelates divalent cations (ions with charges of +2) in the suspension, such as Mg++. This helps break down the cell membrane and inactivate intracellular enzymes. p. 1-12

50. 3. Resuspend the cell pellet in Soln. I Resuspend the bacterial pellet in 200 µl of Solution I by pipeting up and down.