Download Microbiology - Imperial Valley College

Chapter 9
Biotechnology
and
Recombinant DNA
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Lectures prepared by Christine L. Case
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
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Biotechnology and Recombinant DNA
 Vector: Self-replicating DNA used to carry the
desired gene to a new cell
 Clone: Population of cells arising from one cell, each
carries the new gene
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A Typical Genetic Modification
Procedure
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Figure 9.1
A Typical Genetic Modification
Procedure
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Figure 9.1
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Table 9.2
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Table 9.2
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Table 9.3
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
 Destroy bacteriophage DNA in bacterial cells
 Cannot digest (host) DNA with methylated cytosines
ANIMATION: Recombinant DNA Technology
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Table 9.1
Restriction Enzyme & Recombinant
DNA
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Figure 9.2
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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Figure 9.3
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
ANIMATION PCR: Overview
ANIMATION PCR: Components
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PCR
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Figure 9.4
PCR
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Figure 9.4
PCR
ANIMATION PCR: Process
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Figure 9.4
Inserting Foreign DNA into Cells
 DNA can be inserted into a cell by
 Electroporation
 Transformation
 Protoplast fusion
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Figure 9.5b
Process of Protoplast Fusion
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Figure 9.5a
Inserting Foreign DNA into Cells
 DNA can be inserted into a cell by
 Gene gun
 Microinjection
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A Gene Gun
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Figure 9.6
Microinjection of Foreign DNA
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Figure 9.7
Obtaining DNA
 Genomic
libraries are
made of pieces
of an entire
genome stored
in plasmids or
phages
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Figure 9.8
Obtaining DNA
 Complementary
DNA (cDNA) is
made from mRNA
by reverse
transcriptase
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Figure 9.9
Obtaining DNA
 Synthetic DNA is made by a DNA synthesis
machine
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Figure 9.10
Selecting a Clone
Figure 9.11
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Selecting a Clone
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Figure 9.11
Selecting a Clone
Figure 9.12
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Selecting a Clone
Figure 9.12
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Making a Product
E. coli
 Used because it is
easily grown and its
genomics are known
 Need to eliminate
endotoxin from
products
 Cells must be lysed
to get product
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Figure 9.13
Making a Product
Saccharomyces cerevisiae
 Used because it is easily
grown and its genomics
are known
 May express eukaryotic
genes easily
Plant cells and whole plants
 May express eukaryotic
genes easily
 Plants easily grown
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Mammalian cells
 May express
eukaryotic genes
easily
 Harder to grow
Q&A
 Interferons are
species specific, so
that interferons to be
used in humans must
be produced in
human cells. Can you
think of a way to
increase the supply of
interferons so that
they can be used to
treat diseases?
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Therapeutic Applications
 Human enzymes and other proteins
 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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RNA Interference (RNAi)
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Figure 9.14
Random Shotgun Sequencing
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Figure 9.15
The Human Genome Project
 Nucleotides have been sequenced
 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
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Figure 9.17
Southern Blotting
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Figure 9.16
Southern Blotting
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Figure 9.16
Southern Blotting
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Figure 9.16
Forensic Microbiology
 PCR
 Primer for a specific organism will cause application
if that organism is present
 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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Norovirus Outbreak
 Are the outbreaks
related?
 What is the source?
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Clinical Focus, p. 266
Norovirus Outbreak
 RT-PCR with a
norovirus primer
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Clinical Focus, p. 266
Nanotechnology
 Bacteria can make
molecule-sized
particles
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Figure 9.18
Using Agrobacterium
 Bt toxin
 Herbicide
resistance
 Suppression of
genes
 Antisense DNA
 Nutrition
 Human proteins
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Figure 9.19
Using Agrobacterium
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Figure 9.20
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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