microbial biotechnology chapter 5 fall 2006. the structure of microbes prokaryotes archaebacteria...
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Microbial Biotechnology
Chapter 5
Fall 2006
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)
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
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)
Electroporation (figure 5.4) A mixture of bacteria and plasmid are briefly
electrically shocked
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
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)
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!
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)
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
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
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 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?
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)
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)
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?)
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
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
Microbial Diagnostics
(figure 5.16) Using Molecular Techniques to Identify Bacteria RFLP PCR and Real time PCR
PulseNet (Contaminated food)
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