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Group 3
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