Transcript Microbiology

Chapter 9
Biotechnology
and
Recombinant DNA
Copyright © 2010 Pearson Education, Inc.
Lectures prepared by Christine L. Case
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?
Copyright © 2010 Pearson Education, Inc.
Introduction to Biotechnology
Learning Objectives
9-1 Compare and contrast biotechnology, genetic
modification, and recombinant DNA technology.
9-2 Identify the roles of a clone and a vector in
making recombinant DNA.
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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
Check Your Understanding
 Differentiate biotechnology and recombinant DNA
technology. 9-1
 In one sentence, describe how a vector and clone
are used. 9-2
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Tools of Biotechnology
Learning Objectives
9-3 Compare selection and mutation.
9-4 Define restriction enzymes, and outline how they
are used to make recombinant DNA.
9-5 List the four properties of vectors.
9-6 Describe the use of plasmid and viral vectors.
9-7 Outline the steps in PCR, and provide an
example of its use.
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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
 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
Check Your Understanding
 How are selection and mutation used in
biotechnology? 9-3
 What is the value of restriction enzymes in
recombinant DNA technology? 9-4
 What criteria must a vector meet? 9-5
 Why is a vector used in recombinant DNA
technology? 9-6
 For what is each of the following used in PCR:
primer, DNA polymerase, 94°C? 9-7
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Techniques of Genetic Modification
Learning Objectives
9-8 Describe five ways of getting DNA into a cell.
9-9 Describe how a genomic library is made.
9-10 Differentiate cDNA from synthetic DNA.
9-11 Explain how each of the following is used to
locate a clone: antibiotic-resistance genes, DNA
probes, gene products.
9-12 List one advantage of modifying each of the
following: E. coli, Saccharomyces cerevisiae,
mammalian cells, plant cells.
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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?
Copyright © 2010 Pearson Education, Inc.
Check Your Understanding





Contrast the five ways of putting DNA into a cell. 9-8
What is the purpose of a genomic library? 9-9
Why isn’t cDNA synthetic? 9-10
How are recombinant clones identified? 9-11
What types of cells are used for cloning rDNA? 9-12
Copyright © 2010 Pearson Education, Inc.
Applications of rDNA
Learning Objectives
9-13 List at least five applications of rDNA technology.
9-14 Define RNAi.
9-15 Discuss the value of the Human Genome
Project.
9-16 Define the following terms: random shotgun
sequencing, bioinformatics, proteomics.
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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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Check Your Understanding
 Explain how rDNA technology can be used to treat
disease and to prevent disease. 9-13
 What is gene silencing? 9-14
 How are shotgun sequencing, bioinformatics, and
proteomics related to the Human Genome Project?
9-15, 9-16
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Applications of rDNA
Learning Objectives
9-17 Diagram the Southern blotting procedure, and
provide an example of its use.
9-18 Diagram DNA fingerprinting, and provide an
example of its use.
9-19 Outline genetic engineering with Agrobacterium.
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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
Check Your Understanding
 What is Southern blotting? 9-17
 Why do RFLPs result in a DNA fingerprint? 9-18
 Of what value is the plant pathogen Agrobacterium?
9-19
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Safety Issues and Ethics of Using rDNA
Learning Objective
9-20 List the advantages of, and problems associated
with, the use of genetic modification techniques.
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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?
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Check Your Understanding
 Identify two advantages and two problems
associated with genetically modified organisms.
9-20
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