Transcript Microbial Biotechnology

Microbial Biotechnology
Chapter 5
Fall 2008
The Structure of Microbes
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Prokaryotes
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Archaebacteria
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Eubacteria
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Includes halophiles, thermophiles, “extremophiles”
On skin, soil, water, can be pathogenic
Generally smaller than Eukaryotes (1-5um vs 10-100
um)
What are some other characteristics of prokaryotes?
(no nucleus, cell wall (gram stain), binary fission, 20
minute growth rate, conjugation (transfer of DNA by
cytoplasmic bridge), transduction (DNA is packaged
in a virus and infects recipient bacterial cells
Do you know how to isolate single colonies? (Fig 5.2)
Yeast are Important Too!
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Single celled eukaryote
Kingdom: Fungi
Over 1.5 million species –only 10% have been
classified
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
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Microbial Enzymes
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Taq (DNA polymerase), cellulases, proteases,
amylases
Bacterial Transformation (figure 5.3)
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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
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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
Microbial Proteins as Reporters
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Examples: the lux gene which produces
luciferase
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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!
The Yeast Two-Hybrid System
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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)
Fusion protein 1
•Protein of interest linked to
activator (bait)
Fusion protein 2
•Protein of interest linked to
transcription factors (prey)
Using Microbes for a Variety of
Everyday Applications
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Food Products
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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
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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)
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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
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Therapeutic proteins
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Recombinant insulin in bacteria (figure 5.9
and table 5.1)
What is Type I diabetes (insulin-dependent
diabetes mellitus)
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Inadequate production of insulin by beta cells in
the pancreas
Field Applications of Recombinant
Microorganisms
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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!
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Using Microbes Against Other Microbes
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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
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How do antibiotic resistant strains arise?
How can studying bacterial pathogens lead to
new drugs?
Vaccines
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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
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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)
Light chain
Heavy chain
IgA – first line of defense
IgG and IgM – activates
macrophages
Immune Therapy
Used to enhance natural immune responses.
Example - Interferon is a protein that triggers
production of protective proteins (or antibodies) that
inhibit viral replication.
How are Vaccines Made?
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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)
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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
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(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
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(table 5.4)- Examples of medically
important viral genomes that have been
sequenced
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Why?
Decipher genes and their products so that agents
that block attachment, block replication can be
made
Microbial Diagnostics
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(figure 5.16) Using Molecular Techniques
to Identify Bacteria
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RFLP
PCR and Real time PCR
PulseNet
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Used to identify outbreaks
Combating Bioterrorism
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(table 5.5) The use of biological materials as
weapons to harm humans or animals and plants
we depend on for food
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Examples in History
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Throwing plague infected dead bodies over the walls of their
enemies
Using Biotech Against Bioweapons
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Postal service x-raying packages
Antibody tests in the field
PCR tests in the field