Microbiology: Chapter 9

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Microbiology Chapter 9

• Biotechnology and Recombinant DNA

• Biotechnology The use of microorganisms, cells,
  or cell components to make a product.
• Foods, antibiotics, vitamins, enzymes
• Recombinant DNA (rDNA) technology Insertion or
  modification of genes to produce desired proteins

E. coli
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Biotechnology and Recombinant DNA

• Vector Self-replicating DNA used to carry the
  desired gene to a new cell
• A bacterial plasmid or viral genome is used
• Clone Population of cells arising from one cell,
  each carries the new gene or plasmid

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A Typical Genetic Modification Procedure
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A Typical Genetic Modification Procedure
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Selection and Mutation

• Selection Culture a naturally occurring microbe
  that produces desired product
• Mutation Mutagens cause mutations that might
  result in a microbe with a desirable trait
• Site-directed mutagenesis Change a specific DNA
  code to change a protein
• Select and culture microbe with the desired
  mutation

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Restriction Enzymes

• Cut specific sequences of DNA
• Fragments of DNA produced by the same restriction
  enzyme will spontaneously join by base pairing
• DNA ligase can covalently link the DNA
  backbones.
• Destroy bacteriophage DNA in bacterial cells
• Cannot digest (host) DNA with methylated cytosines

ANIMATION Recombinant DNA Technology
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Restriction Enzyme Recombinant DNA
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Vectors

• Carry new DNA to desired cell
• Shuttle vectors can exist in several different
  species
• Plasmids and viruses can be used as vectors

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A Plasmid Vector Used for Cloning
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Polymerase Chain Reaction (PCR)

• To make multiple copies of a piece of DNA
  enzymatically
• Used to
• Clone DNA for recombination
• Amplify DNA to detectable levels
• Sequence DNA
• Diagnose genetic disease
• Detect pathogens

• Can identify an unknown bacterium because the RNA
  primer is specific

ANIMATION PCR Overview
ANIMATION PCR Components
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PCR
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PCR
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PCR
ANIMATION PCR Process
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Inserting Foreign DNA into Cells

• DNA can be inserted into a cell by
• Electroporation a significant increase in the
  electrical conductivity and permeability of the
  cell plasma membrane caused by an externally
  applied electrical field.
• Transformation the transfer of genetic
  information from dead bacteria to live ones
• Protoplast fusion type of genetic modification
  in plants by which two distinct species of plants
  are fused together to form a new hybrid plant
  with the characteristics of both.

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Process of Protoplast Fusion

• Valuable in the genetic manipulation of plant
  algal cells

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Inserting Foreign DNA into Cells

• DNA can be inserted into a cell by
• Gene gun a device for injecting cells with
  genetic information.
• Microinjection process of using a glass
  micropipette to insert substances at a
  microscopic into a single living cell.

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Microinjection of Foreign DNA
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Obtaining DNA

• Genomic libraries are made of pieces of an entire
  genome stored in plasmids, phages, or yeast DNA

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Obtaining DNA

• Complementary DNA (cDNA) is made from mRNA by
  reverse transcriptase
• cDNA is important because it lacks introns

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Obtaining DNA

• Synthetic DNA is made by a DNA synthesis machine

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Selecting a Clone
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Selecting a Clone
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Selecting a Clone
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Selecting a Clone
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DNA Probe

• A single-stranded DNA molecule used in laboratory
  experiments to detect the presence of a
  complementary sequence among a mixture of other
  singled-stranded DNA molecules
• Used to identify bacteria carrying a specific gene

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Making a Product

• E. coli
• Used because it is easily grown and its genomics
  are known
• Its endotoxin eliminate from products
• Cells must be lysed to get product

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Making a Product

• Saccharomyces cerevisiae
• Used because it is easily grown and its genomics
  are known
• May express eukaryotic genes easily

• Mammalian cells
• May express eukaryotic genes easily
• Harder to grow

• Plant cells and whole plants
• May express eukaryotic genes easily
• Plants easily grown

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Therapeutic Applications

• Human enzymes and other proteins (such as human
  insulin)
• Subunit vaccines
• Nonpathogenic viruses carrying genes for
  pathogen's antigens as DNA vaccines
• Gene therapy to replace defective or missing genes

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The Human Genome Project

• Nucleotides have been sequenced in human DNA
• Human Proteome Project may provide diagnostics
  and treatments
• Reverse genetics Block a gene to determine its
  function

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Scientific Applications

• Understanding DNA
• Sequencing organisms' genomes
• DNA fingerprinting for identification (source of
  viral or bacterial pathogens)

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Southern Blotting
Southern blotting can be used to locate a gene in
a cell.
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Southern Blotting
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Southern Blotting
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Forensic Microbiology

• PCR polymerase chain reaction - is a technique
  to amplify a single or few copies of a piece of
  DNA across several orders of magnitude,
  generating thousands to millions of copies of a
  particular DNA sequence.
• Primer for a specific organism will cause
  application if that organism is present

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Forensic Microbiology

• Real-time PCR Newly made DNA tagged with a
  fluorescent dye the levels of fluorescence can
  be measured after every PCR cycle
• Reverse-transcription (RT-PCR) Reverse
  transcriptase makes DNA from viral RNA or mRNA

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Nanotechnology

• Study of the controlling of matter on an atomic
  and molecular scale
• Bacteria can make molecule-sized particles

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Using Agrobacterium

• Uses horizontal gene transfer to cause tumors in
  plants
• Herbicide resistance
• Suppression of genes
• Antisense DNA
• Nutrition
• Human proteins

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Using Agrobacterium
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Gene Therapy

• The correction of a genetic deficiency in a cell
  by the addition of new DNA and its insertion into
  cells or tissues to prevent disease
• In the future, it is hoped that by inserting the
  insulin gene in a diabetic persons pancreatic
  cells, Type I diabetes can be cured

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Safety Issues and Ethics of Using rDNA

• Avoid accidental release
• Genetically modified crops must be safe for
  consumption and for the environment
• Who will have access to an individual's genetic
  information?