1. Biotechnology and Recombinant DNA
Chapter 9
Biotechnology and Recombinant DNA

2. Introduction to Biotechnology
Biotechnology: use of microorganisms, cells, or cell components to make a product
Foods, antibiotics, vaccines, vitamins, enzymes
Recombinant DNA Technology: Insertion or modification of genes to produce desired products (genes or proteins); manufacturing and manipulating genetic material in vitro
also called genetic engineering

3. Overview
Figure 9.1.1

4. Figure 9.1.2

5. Table 9.1.1

6. Table 9.1.2

7. Tools of Biotechnology
(Artificial) Selection: Culture a naturally-occurring microbe that produces desired product
Isolate bacteria and fungi from nature & use pure culture technique
Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait
Site-directed mutagenesis: Change a specific DNA code to change a protein
Select and culture microbe with the desired mutation

8. Tools of Biotechnology
Restriction Enzymes
DNA cutting enzymes that exist in many bacteria
Cut specific sequences of DNA (recognize 4-, 6-, or 8-base sequences), staggered cuts
Destroy bacteriophage DNA in bacterial cells
Cannot digest (host) DNA with methylated cytosines

9. Restriction Enzymes
Figure 9.2

10. Tools of Biotechnology
Vectors
Carry new DNA to desired cell
Plasmids and viruses can be used as vectors
Four properties of vectors
Can self-replicate
Be a size that allows them to be manipulated outside the cell during recombinant DNA procedures
Preservation (circular form of DNA and integrated into host chromosome)
Have a marker within the vector for easy selection

11. Tools of Biotechnology
Shuttle vectors: a plasmid that can exist in several different species
Very useful in the process of genetically modifying multicellular organisms
Viral DNA can usually accept much larger pieces of foreign DNA than plasmid
Retroviruses, adenoviruses, & herpesviruses
Choice of suitable vector depends on many factors (e.g host & size of the DNA to be cloned)

12. Vectors
Figure 9.3

13. Tools of Biotechnology
Polymerase Chain Reaction (PCR)
To make multiple copies of a piece of DNA enzymatically (limited by the choice of primers used)
Cannot be used to amplify an entire genome
Used to
Clone DNA for recombination
Amplify DNA to detectable levels
Sequence DNA
Diagnose genetic disease
Detect pathogens

14. PCR
Figure 9.4.1

15. PCR
Figure 9.4.2

16. Techniques of Genetic Engineering
Inserting foreign DNA into cells
Transformation
Electroporation
Protoplast fusion
Gene gun
Microinjection
Figure 9.5b

17. Techniques of Genetic Engineering
Choice of method is usually determined by the type of vector and host being used
Foreign DNA will survive only if it is either present on a self-replicating vector or incorporated into one of the cell’s chromosomes by recombination

18. Techniques of Genetic Engineering
Transformation: used to insert plasmid vector into a cell
many cell types do not naturally transform need to make them competent (able to take up external DNA)
Electroporation: uses an electrical current to form microscopic pores in the membranes of cells (DNA enter cells through the pores)

19. Techniques of Genetic Engineering
Generally applicable to all cells; ones with cell wall must be converted to protoplasts first
Protoplast fusion: a method of joining two cells by first removing their cell walls
Protoplasts in solution will fuse at a low but significant rate (can add polyethylene glycol to increase the frequency of fusion)
Valuable in the genetic manipulation of plant and algal cells

21. Techniques of Genetic Engineering
Gene gun: Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls
Some of the cells express the introduced DNA as if it were their own if incorporated into host chromosome

22. Techniques of Genetic Engineering
Microinjection: introduce DNA directly into an animal cell using a glass miropipette
Figure 9.6 & 7

23. Biotechnology and Recombinant DNA Part 2
Chapter 9
Biotechnology and Recombinant DNA
Part 2

24. Tools of Biotechnology
Restriction Enzymes
DNA cutting enzymes that exist in many bacteria
Cut specific sequences of DNA (recognize 4-, 6-, or 8-base sequences), staggered cuts
Destroy bacteriophage DNA in bacterial cells
Cannot digest (host) DNA with methylated cytosines

25. Restriction Enzymes
Figure 9.2

26. Tools of Biotechnology
Vectors
Carry new DNA to desired cell
Plasmids and viruses can be used as vectors
Four properties of vectors
Can self-replicate
Be a size that allows them to be manipulated outside the cell during recombinant DNA procedures
Preservation (circular form of DNA and integrated into host chromosome)
Have a marker within the vector for easy selection

27. Tools of Biotechnology
Shuttle vectors: a plasmid that can exist in several different species
Very useful in the process of geneticaly modifying multicellular organisms
Viral DNA can usually accept much larger pieces of foreign DNA than plasmid
Retroviruses, adenoviruses, & herpesviruses
Choice of suitable vector depends on many factors (e.g host & size of the DNA to be cloned)

28. Vectors
Figure 9.3

29. Tools of Biotechnology
Polymerase Chain Reaction (PCR)
To make multiple copies of a piece of DNA enzymatically (limited by the choice of primers used)
Cannot be used to amplify an entire genome
Used to
Clone DNA for recombination
Amplify DNA to detectable levels
Sequence DNA
Diagnose genetic disease
Detect pathogens

30. PCR
Figure 9.4.1

31. PCR
Figure 9.4.2

32. Techniques of Genetic Engineering
Inserting foreign DNA into cells
Transformation
Electroporation
Protoplast fusion
Gene gun
Microinjection
Figure 9.5b

33. Techniques of Genetic Engineering
Choice of method is usually determined by the type of vector and host being used
Foreign DNA will survive only if it is either present on a self-replicating vector or incorporated into one of the cell’s chromosomes by recombination

34. Techniques of Genetic Engineering
Transformation: used to insert plasmid vector into a cell
many cell types do not naturally transform need to make them competent (able to take up external DNA)
Electroporation: uses an electrical current to form microscopic pores in the membranes of cells (DNA enter cells through the pores)

35. Techniques of Genetic Engineering
Generally applicable to all cells; ones with cell wall must be converted to protoplasts first
Protoplast fusion: a method of joining two cells by first removing their cell walls
Protoplasts in solution will fuse at a low but significant rate (can add polyethylene glycol to increase the frequency of fusion)
Valuable in the genetic manipulation of plant and algal cells

36. Fig. 9.5

37. Techniques of Genetic Engineering
Gene gun: Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls
Some of the cells express the introduced DNA as if it were their own if incorporated into host chromosome

38. Techniques of Genetic Engineering
Microinjection: introduce DNA directly into an animal cell using a glass miropipette
Figure 9.6 & 7

39. Obtaining DNA
Gene library: a collection of cloned DNA fragments created by inserting restriction enzyme fragments in a bacterium, yeast, or phage
Make a collection of clones large enough to ensure that at least one clone exists for every gene in the organism
Pieces of an entire genome stored in plasmids or phage

40. Fig. 9.8

41. Obtaining DNA
Cloning genes from eukaryotic organisms poses a special problems due to introns
Need to use a version of the genes that lacks intron = mRNA
cDNA is made from mRNA by reverse transcriptase (mRNA cDNA)
cDNA is the most common method of obtaining eukaryotic genes

42. Fig. 9.9

43. Obtaining DNA Synthetic DNA is made by a DNA synthesis machine
Chain of over 120 nucleotides can be synthesized
Need to know the sequence of the gene
Rare to clone a gene by synthesizing it directly
Plays a much more useful role selection procedures (add desired restriction sites)

44. Selecting a clone
Use antibiotic resistance genes (marker) on plasmid vectors to screen for cells carrying the desired gene (engineered vector)
e.g. Blue-white screening (2 marker genes on the plasmid vector = ampR and -galactosidase)

45. Genetic Engineering
Blue-white screening
Figure

46. Genetic Engineering
Figure

47. Selecting a clone
Need a second procedure to test if screened bacteria does contain desirable genes
Test clones for desired gene product or ID genes itself in the host bacterium
Colony hybridization: use DNA probe that is complementary to the desired genes
DNA probe: short segment of single-stranded DNA that are complementary to the desired gene

48. Colony hybridization
Figure

49. Colony hybridization
Figure

50. Making a gene product
Earliest work in genetic engineering used E. coli to synthesize the gene products
E. coli was used because it is easily grown and its genomics are known
Disadvantages of using E. coli:
Produce endotoxins (Lipid A, part of LPS layer on the cell wall)
Does not secrete protein products need to lyse cells to obtain products
Industry prefers Bacillus subtilis because it secretes their products

51. Making a gene product Use baker’s yeast (Saccharomyces cerevisiae)
Yeast may carry plasmid and has best understood eukaryotic genome
May be more successful in expressing foreign eukaryotic genes than bacteria; likely to secrete products
Use mammalian cells in culture
Hosts for growing viruses (vectors)
Often the best suited to making protein products for medical use (e.g. hormones, cytokines, interferon)

52. Making a gene product Use plant cells in culture
Ti plasmid (from bacterium Agrobacterium tumefaciens), protoplast fusion and gene gun
Use to produce genetically engineered plants
May be sources for plant alkaloids (painkiller), isoprenoids (basis for synthetic rubber), and melanin (for sunscreens)

53. Applications of Genetic Engineering
Produce useful substances more efficiently and cheaper
Obtain information from the cloned DNA that is useful for either basic research or medical applications
Use cloned genes to alter the characteristics of cells or organisms

54. Therapeutic applications
Subunit vaccines
Nonpathogenic viruses carrying genes for pathogen's antigens as vaccines
Gene therapy to replace defective or missing genes
Human Genome Project
Nucleotides have been sequenced
Human Proteome Project may provide diagnostics and treatments

55. Random Shotgun Sequencing
Figure 9.14

56. Scientific Applications
Understanding of DNA
Sequencing organisms' genomes
DNA fingerprinting for identification
Figure 9.16

57. Southern Blotting
Figure

58. Southern Blotting
Figure

59. Southern Blotting
Figure

60. Agricultural Applications
Table 9.2

61. Genetic Engineering Using Agrobacterium
Figure 9.18

62. Safety Issues and Ethics
Avoid accidental release
Genetically modified crops must be safe for consumption and for the environment
Who will have access to an individual's genetic information?

63. Chapter Review: Biotechnology
Use of microorganisms, cells, or cell components to make a product
Organisms that has desirable traits can be obtained by 1) selection (aritificial) or 2) mutation (if you know a specific DNA code you want, you can use site-directed mutagenesis to create a mutant)

64. Biotechnology Restriction enzymes Vectors (plasmids and viruses)
DNA cutting enzymes that cut specific sequence of double-stranded DNA (staggered cuts)
Used to cut out the genes of interest (DNA fragment) and insert the gene into a vector
Vectors (plasmids and viruses)
vehicle to carry new DNA to a desired host cell

65. Biotechnology Vectors
Properties: 1) self-replicating, 2) Be a size that allows them to be manipulated outside the cell (recombinant DNA procedure step), 3) preservation, and 4) have a marker for easy detection
shuttle vectors used to deliver DNA fragments to several different species (esp. useful for multicellular organisms.)

66. Biotechnology Vectors
Virus can accept much larger foreign DNA fragment
Choice of suitable vector depends on the host and the size of the DNA fragment to be cloned

67. Biotechnology Polymerase Chain Reaction (PCR)
To make multiple copies of a DNA fragment; not for amplifying entire genome
Used for 1) cloning DNA for recombination, 2) amplify DNA to detectable levels, 3) DNA sequencing, 4) Diagnose genetic disease, 5) Detect pathogens

68. Genetic Engineering/ Recombinant DNA Technology
Insertion or modification of genes to produce desired products (genes or proteins); manufacturing and manipulating genetic material in vitro
Isolate or obtain DNA with desired gene
Gene library: a collection of cloned DNA fragments created by inserting restriction enzyme fragments in a bacterium, yeast, or phage
In eukaryotes, use cDNA (made from mRNA & reverse transcriptase) instead of DNA
Synthetic DNA

69. Genetic Engineering/ Recombinant DNA Technology
Insert foreign DNA into a host cell
Trasformation: Used to insert plasmid vector into a cell (many cells are not naturally competent to be transformed)
Electroporation: uses an electrical current to form microscopic pores in the membranes of cells & DNA enters cells through the pores (generally applicable to all cells)

70. Genetic Engineering/ Recombinant DNA Technology
Protoplast fusion: a method of joining two cells by first removing their cell walls (valuable for plant and algal cells)
Gene gun: microscopic particles of tungsten or gold coated with DNA and shot through plant cell walls by a burst of helium
Microinjection: Introduce DNA directly into an animal cell using a glass micropipette

71. Genetic Engineering/ Recombinant DNA Technology
Host cells to make a gene product
Earliest work done using E. coli (plasmid vector)
Baker’s yeast (plasmid vector), eukaryotic cells
Mammalian cells in culture (virus vectors, electroporation) --> best suited to make protein products for medical use
Plant cells in culture (Ti plasmid, protoplast fusion, & gene gun) --> genetically engineered plants

72. Genetic Engineering/ Recombinant DNA Technology
Selecting or finding a clone which contains the recombinant vector
Blue-white screening
Colony hybridization

73. Application of Genetic Engineering
Therapeutic application
Subunit vaccines --> no chance of becoming infected from the vaccine
Gene therapy --> replace defective or missing genes with correct genes
Human genome project
may provide diagnostics and treatments

74. Application of Genetic Engineering
Scientific application
Understand DNA; sequence genome; DNA fingerprinting for identifying bacterial/viral pathogens; forensic medicine (paternity, & evidence for a crime)
Agricultural application
Produce genetically engineered plants (more yield per plant, resistant to herbicide, insects, etc.)

75. Chapter Review
Know these terms: biotechnology, genetic engineering/recombinant DNA technology, transformation, electroporation, protoplast fusion, gene gun, and microinjection
Know Fig. 9.1
Know how these tools of biotechnology are used: selection vs. mutation, restriction enzymes, vectors, and PCR (not procedures)

76. Chapter Review
Know the use of gene library, and ways to select a host which has picked up the recombinant (engineered) vector
You do not have to know the procedures for making gene library
Know therapeutic application, human genome project, and agricultural application (from the review slides)