Genetic Engineering Biotechnology

1. Genetic EngineeringBiotechnology

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

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

4. Animal breeding

5. Breeding food plants • “Descendants” of the wild mustard • the “Cabbage family”

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

7. A Brave New World

8. The code is universal • Since all living organisms… • use the same DNA • use the same code book • read their genes the same way

9. TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTTACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT human genome3.2 billion bases

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

11. 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!

12. 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

13. Basic steps in genetic engineering • Isolate the gene • Insert it in a host using a vector • Produce as many copies of the host as possible • Separate and purify the product of the gene

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

15. 5- Transform E. coli with library Each bacteria will receive a single plasmid from the library Each bacteria will grow to form an individual colony Continued “Vibrio DNA library”

16. Tools • DNA you want to clone • Restriction endonucleases (molecular scissors) • Cloning vector (e.g. pGEM, pBR322…) • Ligase enzyme (molecular glue)

17. Step 1: Isolating the gene

18. Step 1: Isolating the gene

19. 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 GTAACG|AATTCACGCTT CATTGCTTAA|GTGCGAA GTAACGAATTCACGCTT CATTGCTTAAGTGCGAA

20. GTAACGAATTCACGCTT CATTGCTTAAGTGCGAA GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA Restriction enzymes • Cut DNA at specific sites • ____________________________ restriction enzyme cut site restriction enzyme cut site

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

22. 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’

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

24. Types of Sticky Ends 5’ overhangs (HindIII) 5’AAGCTT 3’5’A 5’ AGCTT3’ 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’

25. 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

26. TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene sticky ends cut sites chromosome want to add gene to AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA sticky ends stick together chromosome with new gene added TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATCCATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC Sticky ends help glue genes together cut sites gene you want cut sites ________________ DNA molecule DNA ligase joins the strands

27. TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATCCATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGCTAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATCCATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC “new” protein from organism ex: human insulin from bacteria aa aa aa aa aa aa aa aa aa aa How can bacteria read human DNA? Why mix genes together? • Gene produces protein in different organism or different individual human insulin gene in bacteria bacteria human insulin

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

29. Plasmid Vector: pBR322 • First modern cloning vector (1976)

30. pBR322 • Contains: • colE1 origin of replication (ORI)

31. Non-transformed bacteria Bacteria plus plasmid 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)

32. Your favorite DNA EcoRI BamHI Digest with BamH1 and ligate PstI BamHI EcoRI BamHI PstI pBR322 • Contains: 3. A few good restriction sites for inserting foreign DNA BamH1 BamH1 Your favorite DNA

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

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

35. Step 3: inserting vector into host

36. Bacteria • Bacteria are great! • one-celled organisms • reproduce by mitosis • easy to grow, fast to grow • generation every ~20 minutes

37. transformedbacteria gene fromother organism recombinantplasmid cut DNA vector plasmid • 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 + glue DNA

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

39. transformedbacteria gene fromother organism recombinantplasmid + vector plasmid growbacteria harvest (purify)protein Grow bacteria…make more

40. Applications of biotechnology

41. any Questions?