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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. – PowerPoint PPT presentation

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Title: Microbiology: Chapter 9


1
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
2
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

3
A Typical Genetic Modification Procedure
4
A Typical Genetic Modification Procedure
5
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8
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

9
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
10
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11
Restriction Enzyme Recombinant DNA
12
Vectors
  • Carry new DNA to desired cell
  • Shuttle vectors can exist in several different
    species
  • Plasmids and viruses can be used as vectors

13
A Plasmid Vector Used for Cloning
14
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
15
PCR
16
PCR
17
PCR
ANIMATION PCR Process
18
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.

19
Process of Protoplast Fusion
  • Valuable in the genetic manipulation of plant
    algal cells

20
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.

21
Microinjection of Foreign DNA
22
Obtaining DNA
  • Genomic libraries are made of pieces of an entire
    genome stored in plasmids, phages, or yeast DNA

23
Obtaining DNA
  • Complementary DNA (cDNA) is made from mRNA by
    reverse transcriptase
  • cDNA is important because it lacks introns

24
Obtaining DNA
  • Synthetic DNA is made by a DNA synthesis machine

25
Selecting a Clone
26
Selecting a Clone
27
Selecting a Clone
28
Selecting a Clone
29
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

30
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

31
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

32
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

33
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

34
Scientific Applications
  • Understanding DNA
  • Sequencing organisms' genomes
  • DNA fingerprinting for identification (source of
    viral or bacterial pathogens)

35
Southern Blotting
Southern blotting can be used to locate a gene in
a cell.
36
Southern Blotting
37
Southern Blotting
38
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

39
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

40
Nanotechnology
  • Study of the controlling of matter on an atomic
    and molecular scale
  • Bacteria can make molecule-sized particles

41
Using Agrobacterium
  • Uses horizontal gene transfer to cause tumors in
    plants
  • Herbicide resistance
  • Suppression of genes
  • Antisense DNA
  • Nutrition
  • Human proteins

42
Using Agrobacterium
43
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

44
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?
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