2. Genetic Engineering Biotechnology

3. The manipulation of a trait in an organism to create a desired change What is Genetic Engineering?

4. We have been manipulating DNA for generations!  Artificial breeding  creating new breeds of animals & new crop plants to improve our food

5. Animal breeding

6. Breeding food plants  “Descendants” of the wild mustard  the “Cabbage family”

7. Breeding food plants Evolution of modern corn (right) from ancestral teosinte (left).

8. A Brave New World

9. The code is universal  Since all living organisms…  use the same DNA  use the same code book  read their genes the same way

10. TACGCACATTTACGTACGCGGATGCCGCGACT ATGATCACATAGACATGCTGTCAGCTCTAGTAG ACTAGCTGACTCGACTAGCATGATCGATCAGC TACATGCTAGCACACYCGTACATCGATCCTGA CATCGACCTGCTCGTACATGCTACTAGCTACTG ACTCATGATCCAGATCACTGAAACCCTAGATC GGGTACCTATTACAGTACGATCATCCGATCAGA TCATGCTAGTACATCGATCGATACTGCTACTGA TCTAGCTCAATCAAACTCTTTTTGCATCATGAT ACTAGACTAGCTGACTGATCATGACTCTGATCC CGTAGATCGGGTACCTATTACAGTACGATCATC CGATCAGATCATGCTAGTACATCGATCGATACT GCTACTGATCTAGCTCAATCAAACTCTTTTTGC ATCATGATACTAGACTAGCTGACTGATCATGAC TCTGATCCCGTAGATCGGGTACCTATTACAGTA CGATCATCCGATCAGATCATGCTAGTACATCGA TCGATACT human genome 3.2 billion bases

11. Can we mix genes from one creature to another? YES! Green Fluorosceint Protein (GFP)

12. How do we do mix genes?  Genetic engineering  find gene  _______ DNA in both organisms  _______ gene from one creature into other creature’s DNA  _______ new chromosome into organism  organism _______ new gene as if it were its own  organism _______ gene as if it were its own  _____________________________________: Remember: we all use the same genetic code!

13. Uses of genetic engineering  Genetically modified organisms (GMO)  enabling plants to produce new proteins  ___________________________: BT corn  corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)  ___________________________: fishberries  strawberries with an anti-freezing gene from flounder  ___________________________: golden rice  rice producing vitamin A improves nutritional value

14. Basic steps in genetic engineering 1. Isolate the gene 2. Insert it in a host using a vector 3. Produce as many copies of the host as possible 4. Separate and purify the product of the gene

15. Gene Cloning Techniques 1- Grow the target microorganism 2.Extract/isolate DNA DNA target 3- Digest fragment DNA with restriction enzymes 4- Insert DNA fragments in a plasmid cloning vector Recombinant

16. Each bacteria will grow to form an individual colony Continued “Vibrio DNA library” 5- Transform E. coli with library Each bacteria will receive a single plasmid from the library

17. Tools 1. DNA you want to clone 2. Restriction endonucleases (molecular scissors) 3. Cloning vector (e.g. pGEM, pBR322…) 4. Ligase enzyme (molecular glue)

18. Step 1: Isolating the gene

20. Cutting DNA  DNA “scissors”  ____________________________  used by bacteria to cut up DNA of attacking viruses  EcoRI, HindIII, BamHI  cut DNA at specific sites  enzymes look for specific base sequences GTAACGAATTCACGC TT CATTGCTTAAGTGCG AA GTAACG|AATTCACGC TT CATTGCTTAA|GTGCG AA

21. Restriction enzymes  Cut DNA at specific sites  ____________________________ GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA GTAACGAATTCACGC TT CATTGCTTAAGTGCG AA restriction enzyme cut site

22. Sticky ends  Cut other DNA with same enzymes  leave “sticky ends” on both  can glue DNA together at “sticky ends” GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA gene you want GGACCTG AATTCCGGATA CCTGGACTTAA GGCCTAT chromosome want to add gene to GGACCTG AATTCACGCTT CCTGGACTTAA GTGCGAA combined DNA

23. Restriction Endonucleases Restriction endonucleases, a.k.a. “restriction enzymes” or “enzymes” by molecular biologists. Type II restriction enzymes recognize and cut specific DNA sequences 5’-NNNAAGCTTNNN-3’ 3’-NNNTTCGAANNN-5’

24. Example Hind III ( Haemophilus influenza Rd ) –Recognizes: AAGCTT –Cuts in between the two A’s AAGCTT A AGCTT TTCGAA TTCGA A

25. Types of Sticky Ends 5’ overhangs (HindIII) 5’ AAGCTT 3’ 5’ A 5’ AGCTT 3’ 3’ TTCGAA 5’ 3’ TTCGA 5’ A 5’ 3’ overhangs (KpnI) 5’ GGTACC 3’ 5’ GGTAC 3’ C 3’ 3’ CCATGG 5’ 3’ C 3’ CATGG 5’

26. Types of Overhangs  Sticky ends  Examples include HindIII & KpnI  Blunt Ends  Example SmaI  Recognize CCCGGG  Cut between C and G CCCGGG CCC GGG GGGCCC GGG CCC

27. Sticky ends help glue genes together TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCA CGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGT GCGAA gene you wantcut sites AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA chromosome want to add gene tocut sites AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene sticky ends chromosome with new gene added TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACG ATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATG CTAGC sticky ends stick together DNA ligase joins the strands ________________ DNA molecule

28. Why mix genes together? TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACG ATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATG CTAGC  Gene produces protein in different organism or different individual aa “new” protein from organism ex: human insulin from bacteria human insulin gene in bacteria bacteriahuman insulin How can bacteria read human DNA?

29. Step 2: Inserting gene into vector  Vector – molecule of DNA which is used to carry a foreign gene into a host cell

30. Plasmid Vector: pBR322  First modern cloning vector (1976)

31. pBR322 Contains: 1. colE1 origin of replication (ORI)

32. Bacteria plus plasmid Non-transformed bacteria Nutrient media plus antibiotic Overnight growth Only colonies from bacteria that have plasmid pBR322 Contains: 2. Selectable Markers: Ampicillin Resistance (β-lactamase gene) and Tetracycline Resistance (tet gene)

33. pBR322 Contains: 3. A few good restriction sites for inserting foreign DNA PstI Eco RI Bam HI BamH1 Your favorite DNA Digest with BamH 1 and ligate PstI Eco RI Bam HI Your favorite DNA

34. pBR322 Nice Features: √ 200 copies per E. coli cell √ Makes double stranded DNA √ All modern cloning vectors are based on pBR322

35. Advantages over pBR322 1. Makes 1000’s of copies/cell 2. Small size at 2.7 kilobase pairs (kb) = easier uptake by E. coli Next Generation: pUC Plasmids

36. Step 3: inserting vector into host

37. Bacteria  Bacteria are great!  one-celled organisms  reproduce by mitosis  easy to grow, fast to grow  generation every ~20 minutes

38.  A way to get genes into bacteria easily  insert new gene into plasmid  insert plasmid into bacteria  bacteria now expresses new gene  bacteria make new protein + transformed bacteria gene from other organism plasmid cut DNA recombinant plasmid vector glue DNA

39. Blue/White Selection Bacteria plus empty plasmid Non-transformed bacteria Only colonies from bacteria that have plasmid Nutrient media plus antibiotic plus X-Gal Overnight growth Bacteria with plasmid plus insert Colonies with insert - white Colonies w/o insert - blue

40. Grow bacteria…make more grow bacteria harvest (purify) protein transformed bacteria plasmid gene from other organism + recombinant plasmid vector

41. Applications of biotechnology

42. any Questions?