Microbial Biotechnology

1. Microbial Biotechnology Chapter 5 Fall 2006

2. The Structure of Microbes • Prokaryotes • Archaebacteria • Includes halophiles, thermophiles, “extremophiles” • Eubacteria • On skin, pathogens, soil, water • Generally smaller than Eukaryotes (1-5um vs 10-100 um) • What are some other characteristics of prokaryotes? (cell wall (gram stain), no nucleus, binary fission, 20 min growth rate…) • Do you how to isolate single colonies? (Fig 5.2)

3. Yeast are Important Too! • Single celled eukaryote • Kingdom: Fungi • Over 1.5 million species –only 10% have been identified • Source of antibiotics, blood cholesterol lowering drugs • Able to do post translational modifications • Grow anaerobic or aerobic • Examples: Pichia pastoris (grows to a higher density than most laboratory strains), has a no. of strong promoters, can be used in batch processes

4. Microorganisms as Tools • Microbial Enzymes • Taq (DNA polymerase), cellulases, proteases, amylases • Bacterial Transformation (figure 5.3) • The ability of bacteria to take in DNA from their surrounding environment • Cells must be made competent (to take up DNA)

6. Electroporation (figure 5.4) • A mixture of bacteria and plasmid are briefly electrically shocked

8. Cloning and Expression Techniques • Fusion Proteins (Figure 5.5) • Use recombinant DNA methods to insert the gene for a protein of interest into a plasmid containing a gene for a well-known protein that serves as a “tag” • The tag allows for isolation and purification • Ex. His tagged GFP • Affinity chromatography: Ni column binds to repeated his amino acid tag

10. The Yeast Two-Hybrid System • Used to study protein interactions (figure 5.7) • The gene for one protein of interest is cloned and expressed as a fusion protein attached to the DNA-binding domain (DBD) of another gene (the bait). The gene for the second protein of interest is fused to another gene that contains transcriptional activator domain (AD) sequences (prey). If the two proteins interact then transcription occurs and the reporter gene product is expressed! (figure 5.7)

11. Microbial Proteins as Reporters • Examples: the lux gene which produces luciferase • Used to develop a fluorescent bioassay to test for TB (the lux gene is in a virus that only infects M. tuberculosis). If the bacteria is present, the virus infects the cells and the bacterial cells glow!

12. Using Microbes for a Variety of Everyday Applications • Food Products • Rennin used to make curds (solid) and whey in production of cheese • Recombinant rennin is known as chymosin (first recombinant food product approved by FDA)

13. Energy Production in Bacteria • Aerobic respiration (oxygen is final electron acceptor) • Anaerobic respiration (inorganic molecules, such as nitrate, sulfate, or carbonate, are final electron acceptors) • Fermentation/ anaerobic but doesn’t involve an electron transport chain (beers, wines yogurts etc.) Fig 5.8 (done by prokaryotes and eukaryotes) • Purpose: To produce NAD so that the organism can make ATP under anaerobic conditions (substrate level phosphorylation during glycolysis) • Glucose pyruvate (produce ATP and NADH) • Two types: lactic acid and alcohol (NADH NAD) • Pyruvate lactic acid or alcohol and carbon dioxide

16. Therapeutic proteins • Recombinant insulin in bacteria (figure 5.9 and table 5.1) • What is Type I diabetes (insulin-dependent diabetes mellitus) • Inadequate production of insulin by beta cells in the pancreas

18. Field Applications of Recombinant Microorganisms • Ice-minus bacteria (remove ice protein producing genes from P. syringae) • P. fluorescens containing the gene that codes for the bacterial toxin from Bacillus thuringiensis (kills insects) Bt toxin!

19. Using Microbes Against Other Microbes • Antibiotics (table 5.2 and figure 5.10) • Penicillin was the first • Act in a few key ways • Prevent replication • Kill directly • Damage cell wall or prevent its synthesis • How do antibiotic resistant strains arise? • How can studying bacterial pathogens lead to new drugs?

21. Vaccines • Figure 5.11 • First was a vaccine against smallpox (cowpox provides immunity) • DPT-diphtheria, pertussis, and tetanus • MMR –measles, mumps, and rubella • OPV- oral polio vaccine (Sabin)

22. A Primer on Antibodies • Antigen- foreign substances that stimulate an immune response • Types of leukocytes or white blood cells • B-lymphocytes: antibody-mediated immunity • T-lymphocytes: cellular immunity • Macrophages: “cell eating” (phagocytosis)

25. How are Vaccines Made? • They can be part of a pathogen (e.g. a toxin) or whole organism that is dead or alive but attenuated (doesn’t cause disease) • Subunit (toxin) or another part of the pathogen • Attenuated (doesn’t cause disease) • Inactivated (killed) • What about HIV? • What about flu vaccines (why do we have to get a shot every year?)

27. Microbial Genomes • (Figure 5.15 and table 5.3) • Microbial Genome Program (MGP) –the goal is to sequence the entire genomes of microorganisms that have potential applications in environmental, biology, research, industry, and health • Sequencing Strategies

29. Viral Genomes • (table 5.4)- Examples of medically important viral genomes that have been sequenced • Why? Decipher genes and their products so that agents that block attachment, block replication can be made

30. Microbial Diagnostics • (figure 5.16) Using Molecular Techniques to Identify Bacteria • RFLP • PCR and Real time PCR • PulseNet • (Contaminated food)

32. Combating Bioterrorism • (table 5.5) The use of biological materials as weapons to harm humans or animals and plants we depend on for food • Examples in History • Throwing plague infected dead bodies over the walls of their enemies • Using Biotech Against Bioweapons • Postal service x-raying packages • Antibody tests in the field • PCR tests in the field