Foundations in Microbiology

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See how your logic is in figuring this out.
A murderer is condemned to death. He has to
choose between three rooms. The first is full of
raging fires, the second is full of assassins
with loaded guns, and the third is full of lions
that haven't eaten in 3 years. Which room is
safest for him?
The third. Lions that haven't eaten in three
years are dead.
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DNA

• Polymer of nucleotides adenine, thymine,
  cytosine, guanine
• Double helix associated with proteins
• "Backbone" is deoxyribose-phosphate
• Strands held together by hydrogen bonds between
  AT and CG
• Strands are antiparallel

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DNA
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DNA Extraction
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Detergents
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Cold - helps keep the DNA intact during the
extraction process.
Salty - helps the DNA precipitate (solidify and
appear) when alcohol is added.
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Papain - proteases, break apart proteins.
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DNA doesnt dissolve in alcohol. This causes the
DNA to clump together
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DNA
Transcription (RNA polymerase)
RNA
Translation (Ribosomes)
PROTEINS
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Flow of genetic information
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DNA-protein relationship
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Translation
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Mutations changes in the DNA

• Point mutation addition, deletion or
  substitution of a few bases
• Missense mutation causes change in a single
  amino acid
• Nonsense mutation changes a normal codon into a
  stop codon
• Silent mutation alters a base but does not
  change the amino acid

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MutagensIonizing RadiationUV RadiationBreaks
thymine dimersAcridine Orange dyeEthidium
Bromide Intercolators insertion mutation
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Excision repair
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Types of intermicrobial exchange
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conjugation
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transformation
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Generalized transduction
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Transposons DNA segments that shift from one
part of the genome to another
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Genetic engineering

• direct, deliberate modification of an organisms
  genome
• Bioengineering
• Biotechnology use of an organisms biochemical
  and metabolic pathways for industrial production

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I. Tools Techniques of genetic engineering

• enzymes for dicing, splicing, reversing nucleic
  acids
• analysis of DNA

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Enzymes for dicing, splicing, reversing nucleic
acids

• restriction endonucleases recognize specific
  sequences of DNA break bonds
• ligase rejoins bonds cut by endonucleases
• DNA polymerase makes a DNA copy form DNA

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Palindromes
god / dog live/evil star / rats lived / devil
deliver / reviled straw / warts stop / pots
pot / top diaper / repaid war / raw smart /
trams spit / tips
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Analysis of DNA

• Gel electrophoresis- separates DNA fragments
  based on size
• Nucleic acid hybridization probes probes base
  pair with complementary sequences used to detect
  specific sequences
• DNA Sequencing reading the sequence of
  nucleotides in a stretch of DNA
• Polymerase Chain Reaction way to amplify DNA

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Gel ElectrophoresisRestriction Fragment
Length Polymorphism (PFLP)
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Southern blot hybridization
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Fluorescent In-situ Hybridization (FISH)
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DNA sequence technique
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Polymerase chain reaction (PCR)
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http//www.dnalc.org/ddnalc/resources/pcr.html
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Archaebacteria

• The large spring, near Great Fountain Geyser, was
  the source of the culture of Thermus aquaticus
  that is used to make Taq polymerase, a key
  constituent of the polymerase chain reaction. At
  the time of the discovery, this spring was hotter
  than it is today, and its outflow was 70 C.

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Recombinant DNA Technology

• transferring DNA from one organism to another

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Characteristics of cloning vectors

• must be capable of carrying a significant piece
  of donor DNA
• must be readily accepted by the cloning host
• plasmids small, well characterized, easy to
  manipulate can be transferred into appropriate
  host cells through transformation
• bacteriophages have the natural ability to
  inject their DNA into bacterial hosts through
  transduction

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Vector considerations

• origin of replication
• size of donated DNA vector will accept
• gene which confers drug resistance to their
  cloning host

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pBR322
The plasmid pBR322 is one of the most commonly
used E.coli cloning vectors
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Characteristics of cloning hosts

• rapid overturn, fast growth rate
• can be grown in large quantities using ordinary
  culture methods
• nonpathogenic
• genome that is well delineated
• capable of accepting plasmid or bacteriophage
  vectors
• maintains foreign genes through multiple
  generations
• will secrete a high yield of proteins from
  expressed foreign genes

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III. Biochemical Products of Recombinant DNA
Technology

• enables large scale manufacturing of life-saving
  hormones, enzymes, vaccines
• insulin for diabetes
• human growth hormone for dwarfism
• erythropoietin for anemia
• Factor VIII for hemophilia
• HBV vaccine

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IV. Genetically Modified Organisms (GMO)

• Recombinant microbes
• Pseudomonas syringae prevents ice crystals
• Bacillus thuringienisis encodes an insecticide
• Transgenic plants
• Rice that makes beta-carotene
• Tobacco resistant to herbicides
• Peas resistant to weevils
• Transgenic animals
• Mouse models for CF, Alzheimers, sickle cell
  anemia
• Sheep or goats that make medicine in their milk
  semen

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Bioengineering of plants
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Transgenic mice
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Gene therapy

• correct faulty gene in human suffering from
  disease
• ex vivo normal gene is is added to tissues
  taken from the body, then transfected cells are
  reintroduced into the body
• in vivo naked DNA or viral vector is directly
  introduced into patients tissue
• Most trials target cancer, single gene defects
  infections
• Most gene deliveries are carried out by viral
  vectors

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Gene therapy
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Antisense DNA targeting mRNA

• Antisense a nucleic acid strand with a base
  sequence that is complementary to the
  translatable strand
• Antisense DNA gets into the nucleus and binds to
  mRNA, blocking the expression of an unwanted
  protein
• cancers
• Alzheimers disease
• autoimmune diseases

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VI. Genome Analysis

• Gene Mapping
• DNA Fingerprinting
• Microarray analysis

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Gene Mapping

• determining the location of specific genes on the
  chromosomes
• Human Genome Project to determine the
  nucleotide sequence of the gt30,000 genes in the
  genome the importance of these sequences how
  they relate to human disease

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DNA fingerprints
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Pedigree analysis
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Microarray analysis

• Method of determining which genes are actively
  transcribed in a cell under various conditions
• health vs disease
• growth vs differentiation
• could improve accuracy of diagnosis and
  specificity of treatment

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Microarray