Microbiology: Chapter 9

1. Microbiology: Chapter 9 Biotechnology and Recombinant DNA

2. Biotechnology and Recombinant DNA Vector: Self-replicating DNA used to carry the desired gene to a new cell A bacterial plasmid or viral genome is used Clone: Population of cells arising from one cell, each carries the new gene or plasmid

3. A Typical Genetic Modification Procedure

4. A Typical Genetic Modification Procedure

8. 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

9. Restriction Enzymes Cut specific sequences of DNA Fragments of DNA produced by the same restriction enzyme will spontaneously join by base pairing DNA ligase can covalently link the DNA backbones. Destroy bacteriophage DNA in bacterial cells Cannot digest (host) DNA with methylated cytosines

11. Restriction Enzyme & Recombinant DNA

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

13. A Plasmid Vector Used for Cloning

14. 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

15. PCR

16. PCR

17. PCR

18. Inserting Foreign DNA into Cells DNA can be inserted into a cell by Electroporation : a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. Transformation: the transfer of genetic information from dead bacteria to live ones Protoplast fusion: type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both.

19. Process of Protoplast Fusion

20. Inserting Foreign DNA into Cells DNA can be inserted into a cell by Gene gun : a device for injecting cells with genetic information. Microinjection: process of using a glass micropipette to insert substances at a microscopic into a single living cell.

21. Microinjection of Foreign DNA

22. Obtaining DNA Genomic libraries are made of pieces of an entire genome stored in plasmids, phages, or yeast DNA

23. Obtaining DNA Complementary DNA (cDNA) is made from mRNA by reverse transcriptase cDNA is important because it lacks introns

24. Obtaining DNA Synthetic DNA is made by a DNA synthesis machine

25. Selecting a Clone

26. Selecting a Clone

27. Selecting a Clone

28. Selecting a Clone

29. DNA Probe A single-stranded DNA molecule used in laboratory experiments to detect the presence of a complementary sequence among a mixture of other singled-stranded DNA molecules Used to identify bacteria carrying a specific gene

30. Making a Product E. coli Used because it is easily grown and its genomics are known It�s endotoxin eliminate from products Cells must be lysed to get product

31. Making a Product Saccharomyces cerevisiae Used because it is easily grown and its genomics are known May express eukaryotic genes easily Mammalian cells May express eukaryotic genes easily Harder to grow

32. Therapeutic Applications Human enzymes and other proteins (such as human insulin) Subunit vaccines Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines Gene therapy to replace defective or missing genes

33. The Human Genome Project Nucleotides have been sequenced in human DNA Human Proteome Project may provide diagnostics and treatments Reverse genetics: Block a gene to determine its function

34. Scientific Applications Understanding DNA Sequencing organisms' genomes DNA fingerprinting for identification (source of viral or bacterial pathogens)

35. Southern Blotting

36. Southern Blotting

37. Southern Blotting

38. Forensic Microbiology PCR: polymerase chain reaction - is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Primer for a specific organism will cause application if that organism is present

39. Forensic Microbiology 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

40. Nanotechnology Study of the controlling of matter on an atomic and molecular scale Bacteria can make molecule-sized particles

41. Using Agrobacterium Uses horizontal gene transfer to cause tumors in plants Herbicide resistance Suppression of genes Antisense DNA Nutrition Human proteins

42. Using Agrobacterium

43. Gene Therapy The correction of a genetic deficiency in a cell by the addition of new DNA and its insertion into cells or tissues to prevent disease In the future, it is hoped that by inserting the insulin gene in a diabetic person�s pancreatic cells, Type I diabetes can be cured

44. 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?