Microbes come in two main types - prokaryotes and eukaryotes. Prokaryotes like bacteria have no nucleus and reproduce quickly through binary fission. Eukaryotes like yeast are single-celled fungi used for producing antibiotics and in biotechnology. Microbes and their enzymes are useful as tools in cloning, protein expression, and reporting systems. Microorganisms are also used to produce foods and therapeutics through fermentation, and to develop vaccines, diagnostics, and countermeasures against bioweapons.

1. Microbial Biotechnology
Chapter 5

2. The Structure of Microbes
Prokaryotes
• Archaebacteria
• Includes halophiles, thermophiles, “extremophiles”
• Eubacteria
• On skin, soil, water, can be pathogenic

3. The Structure of Microbes
Characteristics of Prokaryotes
• Generally smaller than Eukaryotes
• No nucleus
• Cell wall composed of peptidoglycan
• Conjugation (transfer of DNA by cytoplasmic bridge)
• Transduction (DNA is packaged in a virus and infects
recipient bacterial cells)
• 20 minute growth rate (binary fission)

4. Yeast are Important Too!
Single celled eukaryote
Kingdom: Fungi
Over 1.5 million species
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

5. Microorganisms as Tools
Microbial Enzymes
• Taq (DNA polymerase), cellulases, proteases,
amylases

6. Bacterial Transformation
• The ability of bacteria to
take in DNA from their
surrounding environment
• Bacteria must be made
competent to take up
DNA
Microorganisms as Tools

7. Cloning and Expression Techniques
• Fusion Proteins
Microorganisms as Tools

8. Microbial Proteins as Reporters
• Examples: the lux gene which
produces luciferase
• Used to develop a fluorescent
bioassay to test for TB
Microorganisms as Tools

9. Yeast Two-Hybrid System
• Used to study protein interactions
Microorganisms as Tools

10. 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 ingredient approved by FDA)

11. Using Microbes for a Variety of
Everyday Applications
Food Products
• Energy production in bacteria
• Aerobic or anaerobic

12. Using Microbes for a Variety of
Everyday Applications
Food Products
• Fermentation
(anaerobic respiration)
• Lactic acid fermentation
• Used to make
cheese, yogurt, etc.
• Ethanol fermentation
• Used to make beer
and wine

13. 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!
Using Microbes for a Variety of
Everyday Applications

14. Therapeutic proteins
• Recombinant insulin in bacteria
Using Microbes for a Variety of
Everyday Applications

15.  Using Microbes Against Other Microbes
• Antibiotics
• Act in a few key ways
• Prevent replication
• Kill directly
• Damage cell wall or prevent its synthesis
Using Microbes for a Variety of
Everyday Applications

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

17. 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)
Vaccines

18. Heavy chain
Light chain
IgA – first line of defense
IgG and IgM – activates
macrophages
Vaccines
 Antigens stimulate antibody production in the immune system

19. Vaccines
 Mechanism of Antibody Action

20. 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 flu vaccines (why do we have to get
a shot every year?)
Vaccines

21. Recombinant Vaccines
 Vaccines – provide immunity to infectious microorganisms
Attenuated Vaccine Inactivated Vaccine Subunit Vaccine

22. Recombinant Vaccines
Recombinant Vaccines
• A vaccine produced from a cloned gene
Video: Constructing Vaccines

23. Recombinant Vaccines
DNA vaccines
• Direct injection of
plasmid DNA
containing genes
encoding specific
antigenic proteins

24. Bacterial and Viral Targets
for Vaccines
HIV

25. Microbial Genomes
 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

26. Microbial Genomes
Why study viral genomes?
• Decipher genes and their products so that agents
that block attachment, block replication can be made

27. Microbial Diagnostics
 Using Molecular Techniques to Identify Bacteria
• RFLP
• PCR and Real time PCR
• Sequencing

28. Application: Molecular Epidemiology
• Pulse Net monitors disease outbreaks related to different
strains of food-borne pathogens
Molecular Diagnostics

29. Microbial Diagnostics
Microarrays for tracking
contagious disease
• PulseNet used to identify
outbreaks

30. Combating Bioterrorism
• 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
Microbial Diagnostics

31. Using Biotech Against Bioweapons
• Postal service x-raying packages
• Antibody tests in the field
• PCR tests in the field
• Protein Microarrays for detecting bioweapon pathogens
Microbial Diagnostics