Foundations in Microbiology

1. Foundations in Microbiology PowerPoint to accompany Fifth Edition Talaro Chapter 10 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. Genetic Engineering: A Revolution in Molecular Biology Chapter 10

3. Genetic engineering • direct, deliberate modification of an organism’s genome • bioengineering • Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production

4. I. Tools & Techniques of genetic engineering • enzymes for dicing, splicing, & reversing nucleic acids • analysis of DNA

5. Enzymes for dicing, splicing, & reversing nucleic acids • restriction endonucleases – recognize specific sequences of DNA & break phosphodiester bonds • ligase – rejoins phosphate-sugar bonds cut by endonucleases • reverse transcriptase – makes a DNA copy of RNA - cDNA

7. Analysis of DNA • gel electrophoresis- separates DNA fragments based on size • nucleic acid hybridization & probes – probes base pair with complementary sequences; used to detect specific sequences • DNA Sequencing – reading the sequence of nucleotides in a stretch of DNA • Polymerase Chain Reaction – way to amplify DNA

8. Gel electrophoresis

9. Southern blot hydridization

10. In situ hybridization

11. Sanger DNA sequence technique

12. Polymerase chain reaction (PCR)

13. II. Methods in Recombinant DNA Technology • concerned with transferring DNA from one organism to another • Cloning vectors & hosts • Construction of a recombinant plasmid

15. Characteristics of cloning vectors • must be capable of carrying a significant piece of donor DNA • must be readily accepted by the cloning host • plasmids – small, well characterized, easy to manipulate & can be transferred into appropriate host cells through transformation • bacteriophages – have the natural ability to inject their DNA into bacterial hosts through transduction

16. Vector considerations • origin of replication • size of donated DNA vector will accept • gene which confers drug resistance to their cloning host

17. pBR322

18. Characteristics of cloning hosts • rapid overturn, fast growth rate • can be grown in large quantities using ordinary culture methods • nonpathogenic • genome that is well delineated • capable of accepting plasmid or bacteriophage vectors • maintains foreign genes through multiple generations • will secrete a high yield of proteins from expressed foreign genes

21. III. Biochemical Products of Recombinant DNA Technology • enables large scale manufacturing of life-saving hormones, enzymes, vaccines • insulin for diabetes • human growth hormone for dwarfism • erythropoietin for anemia • Factor VIII for hemophilia • HBV vaccine

22. IV. Genetically Modified Organisms (GMO) • Recombinant microbes • Pseudomonas syringae – prevents ice crystals • Bacillus thuringienisis –encodes an insecticide • Transgenic plants • Rice that makes beta-carotene • Tobacco resistant to herbicides • Peas resistant to weevils • Transgenic animals • Mouse models for CF, Alzheimer’s, sickle cell anemia • Sheep or goats that make medicine in their milk semen

23. Bioengineering of plants

24. Transgenic mice

25. V. Genetic Treatments • Gene therapy • Antisense DNA • Triplex DNA

26. Gene therapy • correct faulty gene in human suffering from disease • ex vivo – normal gene is is added to tissues taken from the body, then transfected cells are reintroduced into the body • in vivo – naked DNA or viral vector is directly introduced into patient’s tissue • Most trials target cancer, single gene defects & infections • Most gene deliveries are carried out by viral vectors

27. Gene therapy

28. Antisense DNA: targeting mRNA • Antisense – a nucleic acid strand with a base sequence that is complementary to the translatable strand • Antisense DNA gets into the nucleus and binds to mRNA, blocking the expression of an unwanted protein • cancers • Alzheimer’s disease • autoimmune diseases

29. Triplex DNA • A triple helix formed when a third strand of DNA inserts into the major groove, making it inaccessible to normal transcription • oligonucleotides have been synthesized to form triplex DNA • oncogenes • viruses • receptor for IL-2

30. Antisense DNA & triplex DNA

31. VI. Genome Analysis • Gene Mapping • DNA Fingerprinting • Microarray analysis

32. Gene Mapping • determining the location of specific genes on the chromosomes • Human Genome Project – to determine the nucleotide sequence of the >30,000 genes in the genome & the importance of these sequences & how they relate to human disease

33. Map of chromosome 16

34. DNA Fingerprinting • Every individual has a unique sequence of DNA • Used to: • identify hereditary relationships • study inheritance of patterns of diseases • study human evolution • identify criminals or victims of disaster

35. DNA fingerprints

36. Pedigree analysis

37. Microarray analysis • Method of determining which genes are actively transcribed in a cell under various conditions • health vs disease • growth vs differentiation • could improve accuracy of diagnosis and specificity of treatment

38. Microarray