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Chapter 9

Chapter 9. Biotechnology and Recombinant DNA. Biotechnology and Recombinant DNA. Biotechnology : The use of microorganisms, cells, or cell components to make a product. Foods, antibiotics, vitamins, enzymes

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Chapter 9

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  1. Chapter 9 Biotechnology and Recombinant DNA

  2. Biotechnology and Recombinant DNA • Biotechnology: The use of microorganisms, cells, or cell components to make a product. • Foods, antibiotics, vitamins, enzymes • Recombinant DNA (rDNA) technology: Insertion or modification of genes to produce desired proteins

  3. Biotechnology and Recombinant DNA • Vector: Self-replicating DNA used to carry the desired gene to a new cell • Clone: Population of cells arising from one cell, each carries the new gene

  4. A Typical Genetic Modification Procedure Figure 9.1

  5. A Typical Genetic Modification Procedure Figure 9.1

  6. Table 9.2

  7. Table 9.2

  8. Table 9.3

  9. Selection and Mutation • Selection: Culture a naturally occurring microbe that produces desired product • 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

  10. Restriction Enzymes • Cut specific sequences of DNA • Destroy bacteriophage DNA in bacterial cells • Cannot digest (host) DNA with methylated cytosines ANIMATION: Recombinant DNA Technology

  11. Table 9.1

  12. Restriction Enzyme & Recombinant DNA Figure 9.2

  13. Vectors • Carry new DNA to desired cell • Shuttle vectors can exist in several different species • Plasmids and viruses can be used as vectors

  14. A Plasmid Vector Used for Cloning Figure 9.3

  15. Polymerase Chain Reaction (PCR) • To make multiple copies of a piece of DNA enzymatically • Used to • Clone DNA for recombination • Amplify DNA to detectable levels • Sequence DNA • Diagnose genetic disease • Detect pathogens PCR

  16. Inserting Foreign DNA into Cells • DNA can be inserted into a cell by • Electroporation • Transformation • Protoplast fusion Figure 9.5b

  17. Process of Protoplast Fusion Figure 9.5a

  18. Inserting Foreign DNA into Cells • DNA can be inserted into a cell by • Gene gun • Microinjection

  19. A Gene Gun Figure 9.6

  20. Microinjection of Foreign DNA Figure 9.7

  21. Selecting a Clone Figure 9.11

  22. Selecting a Clone Figure 9.11

  23. Making a Product E. coli • Used because it is easily grown and its genomics are known • Need to eliminate endotoxin from products • Cells must be lysed to get product Figure 9.13

  24. Mammalian cells May express eukaryotic genes easily Harder to grow Making a Product Saccharomyces cerevisiae • Used because it is easily grown and its genomics are known • May express eukaryotic genes easily Plant cells and whole plants • May express eukaryotic genes easily • Plants easily grown

  25. Therapeutic Applications • Human enzymes and other proteins • Subunit vaccines • Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines • Gene therapy to replace defective or missing genes

  26. The Human Genome Project • Nucleotides have been sequenced, completed in 2003 • Human Proteome Project may provide diagnostics and treatments by providing protein map of entire human body

  27. Scientific Applications • Understanding DNA • Sequencing organisms' genomes • DNA fingerprinting for identification Figure 9.17

  28. Forensic Microbiology • PCR • Primer for a specific organism will cause application if that organism is present • Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle • Reverse-transcription (RT-PCR): Reverse transcriptase makes DNA from viral RNA or mRNA

  29. Norovirus Outbreak • Are the outbreaks related? • What is the source? Clinical Focus, p. 266

  30. Norovirus Outbreak • RT-PCR with a norovirus primer Clinical Focus, p. 266

  31. Nanotechnology • Bacteria can make molecule-sized particles Figure 9.18

  32. Using Agrobacterium • Bt toxin • Herbicide resistance • Suppression of genes • Antisense DNA • Nutrition • Human proteins Figure 9.19

  33. Using Agrobacterium Figure 9.20

  34. Safety Issues and Ethics of Using rDNA • 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?

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