Biotechnology and Genetic Engineering

1. Biotechnology andGenetic Engineering AP Biology Chapter 20

2. Terminology • Genetic engineering – direct manipulation of genetic material for practical purposes • Biotechnology – use of living organisms or their components to make products for us • Recombinant DNA – combining pieces of DNA from different organisms • Gene cloning – making copies of DNA

3. Making recombinant DNA • Plasmids (small circular pieces of DNA in bacterial cells) are used to insert pieces of foreign DNA

4. The DNA is cut using restriction enzymes

5. What are restriction enzymes? • Restriction enzymes come from bacteria and recognize a particular pattern of DNA, often 4, 6 or 8 base pairs long, and then cut the DNA within this recognized sequence. • Bacteria use these enzymes to kill off other competing bacteria by cutting up their DNA.

6. How do they cut? BLUNT ENDS STICKY ENDS

7. ACT GAA TTC CGG AAT GAA TTC TGA CTT AAG GCC TTA CTT AAG Where would the enzyme EcoRI cut?

8. ACT GAA TTC CGG AAT GAA TTC TGA CTT AAG GCC TTA CTT AAG There would be three pieces: one 4 bases, one 12 bases, and one 5 bases.

9. How do bacteria protect it’s own DNA from being cut by the enzymes? It methylates it’s own DNA.

10. Making recombinant DNA in plasmids http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/steps_in_cloning_a_gene.html

11. http://www.nearingzero.net/natural/screenres/natural039.jpg

12. Bacterial plasmids often contain antibiotic resistance genes.

13. Genes can be cloned into vectors such as plasmids

14. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

15. Fig. 20-2a Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 2 Plasmid put intobacterial cell Recombinantbacterium

16. Fig. 20-2b Recombinantbacterium Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest 3 Protein expressedby gene of interest Gene ofInterest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste

17. Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments

18. Fig. 20-4-2 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids

19. Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids

20. Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

21. Steps • Plasmid and DNA of gene of interest are isolated. • Both DNAs are cut with the same restriction enzyme. • “new” DNA is ligated into plasmid • Recombinant plasmids are inserted into bacterial cells. • Plate bacteria on agar. Bacteria will express new genes.

22. Plasmid Maps

24. Plasmid MapsSometimes called restriction maps are graphical representation of plasmids, that show the locations of major identifiable landmarks on DNA like restriction enzyme sites, genes of interest, plasmid length etc.

26. The collection of thousands of clones of bacteria containing recombinant plasmids is called a genomic library.

27. In molecular biology, plasmid (or restriction) maps are used as a reference to engineer plasmids. The plasmids are digested by enzymes chosen and the resulting samples are subsequently run on an electrophoresis gel.

29. Our experiment: to transform E.coli with pGLO plasmid containing the jellyfish gene GFP to make them have the ability to glow

31. To isolate only the cells containing the pGLO DNA, the plasmid contains the beta-lactamase gene which encodes for an ampicillin resistance (Ampr) protein. After the transformation, the cells are grown on a solid medium called an agar plate. This medium will contain the antibiotic ampicillin. In the presence of the ampicillin, only the bacteria containing the pGLO plasmid will have the Ampr protein which will break down the antibiotic, and be able to grow. This process is calledantibiotic selection.

32. GFP results in E.coli

33. This plate shows bacteria expressing six different types of flourescent proteins

34. GRP has been used as tracers to see if the plasmid has been taken up by the bacteria.

35. How much does it cost to make a transgenic mouse? • Transgenic Mouse Production: The current fee for transgenic mouse production for UTMB • investigators is $4000 (for 3 days of injections into [C57BL/6 X C3H/He]F2 embryos) or $5200 • (for 4 days of injection into C57BL/6 or FVB/N embryos; $1250 for each additional day

36. Storing Cloned Genes in DNA Libraries • Plasmid libraries containing genes of interest cloned in • Phage library that is made using bacteriophages which store genes of interest

37. Fig. 20-5a Foreign genomecut up withrestrictionenzyme or Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones (a) Plasmid library (b) Phage library

38. Viruses used as vectors

39. BACs (bacterial artificial chromosome) are another type of vector used in DNA library construction • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert

40. Fig. 20-5 Foreign genomecut up withrestrictionenzyme Large insertwith many genes Large plasmid or BACclone Recombinantphage DNA Bacterial clones Recombinantplasmids Phageclones (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

41. A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell • A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/cdna.html

42. Fig. 20-6-1 DNA innucleus mRNAs in cytoplasm

43. Fig. 20-6-2 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand

44. Fig. 20-6-3 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA

45. Fig. 20-6-4 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA DNA polymerase

46. Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Primer DNAstrand DegradedmRNA http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/fish.html DNA polymerase cDNA

47. Ways to introduce new genes into bacteria. Conjugation – through tubes between bacteria Transformation – negative DNA taken up Transduction by bacteriophages or other viruses Mutation ALL of these introduce GENETIC VARIATION!

48. Nucleic Acid Hybridization • Used to detect genes • The DNA of the cell is denatured to produce single stranded DNA. • The radioactive probe will hybridize (bond) with complementary bases if present. • Probes can be radioactive isotopes or flourescent dyes.

50. The radioactive probe is made by determining a short segment of the protein sequence, then "back translating" to the possible short DNA sequences called oligomers. Then these DNA oligomers (known as "oligos") are radiolabeled, and applied to the blotted clones.  They should hybridize only to clones containing sequence encoding the desired protein.