Microbial Biotechnology

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Chapter 5

• Microbial Biotechnology

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Interesting Facts

• Microbes have existed on the earth for over 3.5
  billion years
• 50 of the living matter is comprised of
  micoorganisms
• Less than 1 of all bacteria have been
  identified, cultivated and studied in the
  laboratory
• Yet we are literally surrounded by microbes all
  the time

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What bacteria look like
spheres or cocci growing in chains
cork-screw
rods
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How we cultivate bacteria
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Microorganisms as tools for biotechnology

• Ways of getting foreign genes into a bacterial
  cell
• Transformation
• Naked DNA taken up by cell from the environment
• Electroporation
• Electric shock opens up the cell wall and allows
  DNA to enter the cell from the environment

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Transformation
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E. coli bacterium transformed with a gene from a
jellyfish
The jellyfish gene encodes a green fluorescent
protein (GFP) which allows you to easily see a
bacterial cell that has been transformed and
expresses the protein In this case, the gene is
referred to as a reporter gene because it is
reporting on the location of the bacterium.
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Electroporation
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Cloning and Expression Techniques

• Bacterial fusion or hybrid proteins for synthesis
  and isolation of recombinant proteins
• use recombinant DNA method 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 for the protein of interest
• the tag protein then allows for the isolation and
  purification of the recombinant protein as a
  fusion protein

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Expression vectors

• Plasmid vectors for making fusion proteins are
  called expression vectors because they enable
  bacterial cells to produce or express large
  amounts of protein
• vectors have gene encoding
• Maltose-binding protein

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Fusion Proteins-how they are made and recovered
Recover from culture medium and purify
DNA
peptide
protein of interest
mRNA
Maltose-binding protein
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Fusion Proteins
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Microbial Proteins as Reporters
Luciferase (lux gene)
Light organ filled with bacterium Vibrio fisheri
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Fermentation Products
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Ethanol fermentation

• Anaerobic reactions
• Conversion of sugar from grains and fruits to
  EtOH
• grains beer
• grapes wine and vinegar
• By manipulating rate of biochemical reaction in
  the culture, brewers can control the EtOH
  content.
• Ethanol fermentation microorganism
• Saccharomyces cervisiae
• (yeast fungi)

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Fermentation reactions
Many enzyme proteins required for the conversion
of glucose to ethanol
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Same yeast used to make EtOH is used to make
bread

• Yeast uses sugar in dough to make EtOH and CO2
• CO2 gets trapped in dough and causes it to
  rise.
• Baking the dough causes to gas to escape, leaving
  holes behind in the bread.
• Cooking the bread completely causes all the EtOH
  to evaporate
• sourdough bread has some of the EtOH retained in
  the bread because it is undercooked.

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Fermentation-anaerobic metabolism
stopped

• Lactic acid fermentation
• Lactococcus lactis (bacterium)
• Used to make
• sauerkraut from cabbage
• yogart
• vinegar
• citric acid in fruit
• methanol and acetone

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Therapeutic proteins

• Insulin is part of a class of proteins called
  hormones
• It is produced by cells in our pancreas and
  secreted into the bloodstream to stimulate uptake
  of blood glucose into body cells such as muscle
  tissue
• Allowing blood glucose levels to remain high
  causes health problems
• high blood pressure
• poor blood circulation
• cataracts

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• Human insulin consist of 2 polypeptides
• A subunit (21 amino acids)
• B subunit (30 amino acids)
• The 2 peptides bind to each other by reversible
  chemical bonds
• Pancreas cells produce the 2 subunits as a single
  polypeptide chain and then enzymatically cut the
  polypeptide to produce the 2 subunits, which then
  fold properly into an active insulin protein.

Enzymatic cleavage in cells of body
Functionally-active hormone
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Recombinant insulin
b-gal
antibody
Lactose in culture medium
insulin subunit
Combine genes to make fusion protein
bead
b-galactosidase protein
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Therapeutic Proteins from Recombinant Bacteria
Application cystic fibrosis treat different
cancers leukemia minimize tissue damage after
heart attack

• Function
• digests DNA
• stimulates cell growth
• binds and destroys harmful free radicals

Protein

• Protein DNase
• Interferons and Interleukins
• Superoxide dismutase

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Target sites of antimicrobials (antibiotics) in
bacterial cells
erythromycin tetracycline
pennicilin
ciprofloxacin
fostriencin
rifampicin
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Vaccines

• First vaccine developed by Edward Jenner in 1796
• used a live cowpox virus to vaccinate humans
  (himself) against smallpox
• based on claims that milkmaids exposed to cowpox
  virus in udder infections on cow never got
  smallpox disease.
• Exposure to cowpox fluid stimulated immune system
  of human volunteers to develop protection against
  smallpox

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Vaccines

• Today, DPT vaccine is given to infants to protect
  them from
• diptheria toxin
• pertussis toxin
• tetanus toxin
• Another common vaccine is MMR
• measles
• mumps
• rubella
• Polio vaccine

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Benchmarks in disease control
stopped
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Polio vaccine

• Many people dont have their children immunized
  these days because they think that polio doesnt
  exist or pose a real threat now.
• Virus is still around and children can still
  succumb to the disease.
• Question Is the risk of developing a side-effect
  or getting the disease from the vaccine worth the
  risk of avoiding the disease when exposed to a
  natural source of the virus?

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Normal Immune Response
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• Primary response is slow and may not be able to
  produce enough antibody-producing cells to
  destroy antigen if it is being produced by
  bacteria replicating in our bodies
• Secondary response is much quicker, so there is a
  better chance of destroying antigen and bringing
  disease under control

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Mechanisms of Antibody Action
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How do vaccines work?

• Vaccines induce the primary response without
  causing the disease so that when an individual is
  later exposed to the real disease-causing agent,
  a secondary response will occur and provide a
  more rapid and effective immunological response
  to neutralize the disease-causing agent.

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Types of vaccines

• Inactivated vaccines - killed virus)
• polio
• Attenuated vaccines - live virus but genetically
  engineered so as not to replicate in host
• polio
• Subunit vaccines - portion of infectious agent
  that elicits good antibody response (protein or
  lipid molecule)
• hepatitis B virus protein

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Current efforts to develop vaccines against
emerging diseases

• HIV
• 33 million people affected
• high mutation rate of HIV virus compromises
  efficacy of vaccines developed to date

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HIV life cycle
RNA virus
Receptor protein on T-cell
RNA?DNA
T-cell is a type of white blood cell that
participates in cell mediated immunity
T-cell
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Current efforts to develop vaccines against
emerging diseases

• Tuberculosis
• 3000-year-old disease
• 2-3 million deaths/year 7th leading cause of
  death
• current treatment regime is demanding 4 drugs
  taken daily for 6-18 months patients dont
  complete regime
• new bacterial strains are resistant to
  antibiotics that controlled previous strains
• genome of Mycobacterium tuberculosis has been
  sequenced and new proteins have been discovered
  that are good candidates for developing vaccines
  against
• TB has become the single largest cause of death
  among AIDS patients

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How Human Genome Project has been used to fight
human disease

• Tools and methods developed to sequence human
  genome are now being used to sequence genomes of
  bacteria that cause disease
• New proteins located on the bacterial cell
  surface were discovered by sequencing
  Streptococcus pneumoniae genome
• These are now used as target antigens for subunit
  vaccines

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Sequencing a microbial genome
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beginning
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Using microbial genome information to identify
causative agents of outbreaks of disease

• E. coli strain 0157h7 produces a lethal toxin
  that causes 20,000 cases of food poisoning each
  year.
• The gene sequence for the toxin is known
• Strains recovered from patients producing the
  toxin can be detected and distinguished from
  harmless strains using polymerase chain reaction
  (PCR)

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• The USDA has set up labs (PulseNet) across the
  country to do rapid identification of bacteria
  using rapid molecular approaches
• What methods did we use to identify causative
  agents of disease before we could easily test for
  specific genes?
• Why are the new rapid methods preferred for this
  application?

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Microbes as bioweapons

• Bioterrorism is not new in this country.
• In 1800s the U.S. military distributed blankets
  and other articles contaminated with smallpox
  virus to the Native Americans who were occupying
  land in the West that settlers wanted to take
  possession of.
• Since the Native Americans had never been exposed
  to this virus, they had virtually no resistance
  to the disease.
• The American military exploited this situation to
  debilitate as many Native Americans as possible.

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• So many Native Americans became infected with and
  died from exposure to smallpox that they were
  unable to hold on to their hunting grounds and
  tribal sites and the survivors were relegated to
  reservations.
• Americans were terrorized after 9-11-01 when
  spores of anthrax bacteria were transmitted in
  the mail and 5 people died.
• Anthrax bacteria were found in large-scale
  production in Tokyo in 2000 and being used to
  attempt to kill people in that city.

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• No evidence of any deaths from the release in
  Tokyo. Why?
• Disease-causing strain carries 2 plasmids each
  containing a different toxin gene.
• Both genes must be expressed to cause disease.

Toxin gene 2
Bacillus anthracis
Toxin gene 1
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• Strain produced and disseminated by terrorists in
  Toyko carried only one of the plasmids, so it was
  not pathogenic.
• Bioterrorists are not particularly knowledgeable
  in the molecular biology of disease.
• However, with the appropriate knowledge such as
  that which you have acquired in this course, a
  person could produce and disseminate a
  disease-causing microbe into the environment.
• Safeguards now in place to restrict public access
  to critical research results

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What would be an effective way to use pathogenic
microbes to intentionally kill a large number of
Americans or American allies?

• Choice of agent?
• Delivery system?
• Dispersion mechanism?
• Targets?

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Potential Biological Weapons

• Brucella
• Bacillus anthrasis
• Clostridium botulinum
• Ebola or Marburg virus
• Francisella tularensis
• Influenza virus
• Rickettsia
• Variola virus (smallpox)
• Yersinia pestis (plague)

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Food pathogens as a target of bioterrorism

• Foot-and-mouth disease (virus)
• Africa swine fever virus
• Stem rust fungus for cereal crops
• Southern corn leaf blight (fungus)
• Rice blast (fungus)
• Potato blight virus

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How could U.S. mount an effective response to
bioweapons released in this country?

• Use of biotechnology to detect bioweapons
  released into the environment
• similar strategy as one currently used to detect
  and control infectious disease outbreaks that
  occur naturally?
• Air, water and soil monitoring
• rapid-response teams and programs
• new strategies needed
• sanitizing mail uv or x-ray treatment before
  handling and distribution

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Protein Microarray for Detecting Bioweapon
Pathogens