Chapter 5. Microbial Biotechnology. PowerPoint Lectures for Introduction to Biotechnology, Second Edition William J.Thieman and Michael A.

Transcription

1 PowerPoint Lectures for Introduction to Biotechnology, Second Edition William J.Thieman and Michael A.Palladino Chapter 5 Microbial Biotechnology Lectures by Lara Dowland

2 Chapter Contents 5.1 The Structure of Microbes 5.2 Microorganisms as Tools 5.3 Using Microbes for a Variety of Everyday Applications 5.4 Vaccines 5.5 Microbial Genomes 5.6 Microbes for Making Biofuels 5.7 Microbial Diagnostics 5.8 Combating Bioterrorism

3 5.1 The Structure of Microbes Microbes (microorganisms) are tiny organisms that are too small to be seen individually by the naked eye and must be viewed with the help of a microscope Bacteria, fungi, algae, and protozoa

4 5.1 The Structure of Microbes Structural Features of Bacteria Small (1 5 µm) No nucleus; DNA is contained in a single, circular chromosome May contain plasmids Cell wall that surrounds plasma membrane contains peptidoglycan; provides rigidity for protection Some bacteria contain an outer layer of carbohydrates in a structure called a capsule

5 5.1 The Structure of Microbes

6 5.1 The Structure of Microbes Bacteria are classified by the Gram stain Gram + bacteria stain purple Have simple cell walls rich in peptidoglycan Gram bacteria stain pink Have complex cell wall structures with less peptidoglycan

7 G(+) vs. G(-) bacteria

8 Peptidoglycan

9 5.1 The Structure of Microbes Bacteria vary in size and shape Most common shapes Cocci spherical cells Bacilli rod-shaped cells Spiral corkscrew-shaped cells

10 5.1 The Structure of Microbes Single, circular chromosome is relatively small 2 4 million base pairs Some bacteria contain plasmids as well Plasmids often contain genes for antibiotic resistance and genes encoding proteins that form connecting tubes called pili Plasmids are an essential tool for biotechnology

11 5.1 The Structure of Microbes Bacteria grow and divide rapidly Divide every 20 minutes or so Millions of cells can be grown on small dishes of agar or in liquid culture media Easy-to-make mutant strains to be used for molecular and genetic studies

12 5.1 The Structure of Microbes

13 5.1 The Structure of Microbes Yeast single-celled eukaryotic microbes; fungi Sources of antibiotics and drugs that lower cholesterol Mechanisms of gene expression resemble those in human cells

14 5.1 The Structure of Microbes Yeast Can grow in the presence of oxygen (aerobic) or in the absence of oxygen (anaerobic) Pichia pastoris Grows to a higher density in liquid culture than other yeast strains Has a number of strong promotors that can be used for production of proteins Can be used in batch processes to produce large number of cells

15 5.2 Microorganisms as Tools Microbial Enzymes Used in applications from food production to molecular biology research Taq (& Pfu) DNA polymerase Isolated from a thermophile Cellulase Makes animal food more easily digestible Stone-washed jeans Subtilisin Laundry detergents

16 5.2 Microorganisms as Tools Transformation the ability of bacteria to take in DNA from their surrounding environment Essential step in the recombinant DNA cloning process Competent cells are cells that have been treated so they are ready to take up DNA easily Treat cells with ice-cold solution of calcium chloride

17 5.2 Microorganisms as Tools Transformation Target DNA is introduced into a plasmid containing one or more antibiotic resistance genes Plasmid vector is mixed in a tube with competent cells and placed on ice Cells are heated briefly (heat shock) to allow DNA to enter cell Grow in liquid media Plate on agar plates containing antibiotics

18 5.2 Microorganisms as Tools

19 5.2 Microorganisms as Tools Electroporation An instrument called an electroporator produces a brief electrical shock that introduces DNA into the cells without killing them Advantages Rapid Requires fewer cells Can be used to introduce DNA into other cell types More efficient process

20 5.2 Microorganisms as Tools

21 5.2 Microorganisms as Tools Bacteria can be used to mass-produce proteins Bacterial fusion proteins Gene for protein of interest is inserted into a plasmid containing a gene for a well-known protein that serves as a tag The tag protein allows for the isolation and purification of the recombinant protein as a fusion protein Plasmid vectors used are often called expression vectors Incorporate prokaryotic promoter sequences

22 5.2 Microorganisms as Tools

23 5.2 Microorganisms as Tools Microbial Proteins as Reporters Bioluminescence method of producing light used by marine organisms Created by bacteria such a Vibrio fisheri that use marine organism as a host Create light through action of lux genes

24 5.2 Microorganisms as Tools Microbial Proteins as Reporters Lux genes have been cloned and used to study gene expression Clone lux genes into plasmid If inserted into animal or plant cells, will produce luciferase and will fluoresce, providing a visual indicator of gene expression

25 5.3 Using Microbes for a Variety of Everyday Applications Food Products Breads, yogurts, cheeses, sauerkraut Beer, wines, champagnes, liquors Fermentation process of deriving energy from sugars in the absence of oxygen Lactic acid fermentation Alcohol fermentation

26 5.3 Using Microbes for a Variety of Everyday Applications

27 5.3 Using Microbes for a Variety of Everyday Applications Therapeutic Proteins Bacteria are used to produce medically important proteins. For example, insulin.

28 5.3 Using Microbes for a Variety of Everyday Applications

29 5.3 Using Microbes for a Variety of Everyday Applications Antibiotics Produced by microbes that inhibit the growth of other microbes 1928 discovery of penicillin by Alexander Fleming Majority are produced by bacteria, and inhibit the growth of other bacteria

30 5.3 Using Microbes for a Variety of Everyday Applications

31 5.3 Using Microbes for a Variety of Everyday Applications

32 5.3 Using Microbes for a Variety of Everyday Applications Field Applications Degradation of waste products Bioremediation of polluted environments 1987 Steven Lindow at University of California First field application Creation of ice-minus bacteria would provide protection of crops from frost Genetically altered strain of bacteria that would protect plants against root-eating insects on cotton and corn

33 5.4 Vaccines First vaccine developed in 1796 by Edward Jenner Used live cowpox virus to vaccinate against smallpox

34 5.4 Vaccines Immune System and Antibodies Antigens are foreign substances that stimulate an immune response Whole bacteria, fungi, and viruses Proteins, lipids, or carbohydrates Immune system responds to antigens by producing antibodies Called antibody-mediated immunity B cells, with the help of T cells, recognize and bind to the antigen B cells then develop to form plasma cells that produce antibodies

35 5.4 Vaccines Immune System and Antibodies Antibodies are very specific Bind to the antigen Macrophage can then recognize the antigens coated with antibodies and eat them Sometimes our natural production of antibodies is not enough to protect us from pathogens

36 Antigens stimulate antibody production by the immune system

37 Mechanisms of antibody action

38 5.4 Vaccines Vaccines parts of a pathogen or whole organisms that can be given to humans or animals by mouth or by injection to stimulate the immune system against infection by those pathogens

39 5.4 Vaccines Four Major Strategies to Make Vaccines Subunit vaccines are made by injecting portions of viral or bacterial structures Attenuated vaccines use live bacteria or viruses that have been weakened through aging or by altering their growth conditions to prevent replication Inactivated (killed) vaccines are made by killing the pathogen and using the dead or inactivated microorganism for the vaccine DNA-based vaccines have been attempted but so far they have not proven to be widely effective.

40 5.4 Vaccines Bacterial and Viral Targets Influenza: avian flu (H5N1), swine flu (H1N1) Tuberculosis (TB) Malaria HIV

41 5.4 Vaccines

42 5.5 Microbial Genomes 1994 Microbial Genome Program (MGP) To sequence the entire genomes of microorganisms that have potential applications in environmental biology, research, industry, and health as well as genomes of protozoan pathogens 2008 NIH announced plans for the Human Microbiome Project 5-year project to sequence 600 genomes of microorganisms that live on and inside humans

43 5.5 Microbial Genomes Why sequence microbial genomes? Streptococcus pneumoniae, which causes ear and lung infections, kills 3 million children worldwide each year Many of the vaccines are ineffective in children In 2001 the genome was sequenced and many genes encoding proteins on the surface of the bacteria were discovered Could lead to new treatments, including gene therapy

44 5.5 Microbial Genomes Why sequence microbial genomes? Identify genes involved in bacterial cell metabolism, cell division, and genes that cause human and animal illnesses

45 5.5 Microbial Genomes

46 5.7 Microbial Diagnostics Microbial Diagnostics techniques used to detect and track microbes Bacterial Detection Strategies RFLP analysis, PCR and DNA sequencing Databases are available for comparison of clinical samples Used to detect and track bacterial contamination of food

47 Using molecular techniques to identify bacteria

48 Hot-response gene expression signatures for pathogen identification

49 5.8 Combating Bioterrorism Bioterrorism the use of biological materials as weapons to harm humans or the animals and plants we depend on for food Only 12 or so organisms could feasibly be cultured, refined, and used in bioterrorism Delivered by aerosols, crop duster planes, or water supplies

50 5.8 Combating Bioterrorism

51 Protein microarray for detecting bioweapon pathogens

52 Battling Bioterrorism