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Microbial Biotechnology

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Chapter 5 Microbial Biotechnology Interesting Facts Microbes have existed on the earth for over 3.5 billion years 50% of the living matter is comprised of ... – PowerPoint PPT presentation

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Title: Microbial Biotechnology


1
Chapter 5
  • Microbial Biotechnology

2
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

3
What bacteria look like
spheres or cocci growing in chains
cork-screw
rods
4
How we cultivate bacteria
5
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

6
Transformation
7
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.
8
Electroporation
9
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

10
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

11
Fusion Proteins-how they are made and recovered
Recover from culture medium and purify
DNA
peptide
protein of interest
mRNA
Maltose-binding protein
12
Fusion Proteins
13
Microbial Proteins as Reporters
Luciferase (lux gene)
Light organ filled with bacterium Vibrio fisheri
14
Fermentation Products
15
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)

16
Fermentation reactions
Many enzyme proteins required for the conversion
of glucose to ethanol
17
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.

18
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

19
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

20
  • 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
21
Recombinant insulin
b-gal
antibody
Lactose in culture medium
insulin subunit
Combine genes to make fusion protein
bead
b-galactosidase protein
22
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

23
Target sites of antimicrobials (antibiotics) in
bacterial cells
erythromycin tetracycline
pennicilin
ciprofloxacin
fostriencin
rifampicin
24
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

25
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

26
Benchmarks in disease control
stopped
27
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?

28
Normal Immune Response
29
(No Transcript)
30
  • 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

31
Mechanisms of Antibody Action
32
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.

33
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

34
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

35
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
36
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

37
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

38
Sequencing a microbial genome
39
beginning
40
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)

41
  • 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?

42
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.

43
  • 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.

44
  • 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
45
  • 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

46
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?

47
Potential Biological Weapons
  • Brucella
  • Bacillus anthrasis
  • Clostridium botulinum
  • Ebola or Marburg virus
  • Francisella tularensis
  • Influenza virus
  • Rickettsia
  • Variola virus (smallpox)
  • Yersinia pestis (plague)

48
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

49
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

50
Protein Microarray for Detecting Bioweapon
Pathogens
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