Transcript ch_11_lecture_presentation_PCx

PowerPoint® Lecture
Presentations prepared by
John Zamora
Middle Tennessee State
University
CHAPTER
11
Genetic
Engineering
and
Biotechnology
© 2015 Pearson Education, Inc.
11.1 Restriction Enzymes and Nucleic Acid
Separation
• Genetic engineering: using in vitro techniques to
alter genetic material in the laboratory
• Basic techniques include:
• Restriction enzymes
• Gel electrophoresis
• Nucleic acid hybridization
• Nucleic acid probes
• Molecular cloning
• Cloning vectors
© 2015 Pearson Education, Inc.
11.1 Restriction Enzymes and Nucleic Acid
Separation
• Restriction enzymes: recognize specific DNA
sequences and cut DNA at those sites
• Widespread among prokaryotes
• Defense against foreign DNA
• Rare in eukaryotes
• Essential molecular tools for in vitro DNA manipulation
© 2015 Pearson Education, Inc.
11.1 Restriction Enzymes and Nucleic Acid
Separation
• Three classes of restriction enzymes
• Type II cleave DNA within their recognition sequence
and are most useful for specific DNA manipulation
• Restriction enzymes recognize palindromes (inverted
repeat sequences)
• Typically 4–8 base pairs long
RACECAR
• Sticky ends or blunt ends
EYE
MADAM
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11.1 Restriction Enzymes and Nucleic Acid
Separation
© 2015 Pearson Education, Inc.
Figure 11.1a
11.1 Restriction Enzymes and Nucleic Acid
Separation
• Modification enzymes: protect cell's DNA for
restriction enzymes
• Chemically modify nucleotides in restriction recognition
sequence
• Modification generally consists of methylation of DNA
© 2015 Pearson Education, Inc.
Figure 11.1b
11.1 Restriction Enzymes and Nucleic Acid
Separation
• Gel electrophoresis: separates DNA molecules
based on size
• Electrophoresis uses an electrical field to separate
charged molecules
• Gels are usually made of agarose, a polysaccharide
• Nucleic acids migrate through gel toward the positive
electrode because of their negatively charged
phosphate groups
• Gels can be stained (e.g. ethidium bromide) so
DNA can be visualized under UV light
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11.1 Restriction Enzymes and Nucleic Acid
Separation
© 2015 Pearson Education, Inc.
Figure 11.2a
Figure 11.2b
11.1 Restriction Enzymes and Nucleic Acid
Separation
• The same DNA that has been cut
with different restriction enzymes
will result in different banding
patterns on an agarose gel
• Size of fragments can be
determined by comparison to a
standard called a DNA ladder
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Figure 11.2b
11.2 Nucleic Acid Hybridization
Southern blot (DNA)
• Nucleic acid hybridization:
base pairing of single strands
of DNA or RNA from two
different sources to give a
hybrid double helix
• Segment of single-stranded
DNA that is used in
hybridization and has a
predetermined identity is called
a nucleic acid probe
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Northern blot (DNA)
Figure 11.3
11.2 Nucleic Acid Hybridization
• FISH: Fluorescent In Situ Hybridization
• Uses fluorescent probe attached to oligonucleotide
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Figure 11.4
11.3 The Polymerase Chain Reaction
• The polymerase chain reaction (PCR) is basically
DNA replication in a test tube
• Kary Mullis
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11.3 The Polymerase Chain Reaction
• The polymerase chain reaction (PCR) is basically
DNA replication in a test tube
• Also called DNA amplification, product = amplicon
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Figure 11.5
11.3 The Polymerase Chain Reaction
• Variations of PCR
• Reverse transcription PCR (RT PCR) (Fig. 11.6)
• Can make DNA from an RNA template
• Uses the enzyme reverse transcriptase
• Quantitative PCR (q PCR)
• Uses fluorescent probe to monitor the amplification
process
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11.4 Essentials of Molecular Cloning
• Molecular cloning:
isolation and
incorporation of a
piece of DNA into
a vector so it can
be replicated and
manipulated
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Figure 11.7
11.4 Essentials of Molecular Cloning
• Essential to detect the correct clone
• Initial screen: antibiotic resistance,
plaque formation
• If cells express the foreign gene
and its expression can be detected,
then screening is relatively easy
• Nucleic acid probes/PCR – if gene is
not expressed
• Antibodies – if gene is expressed
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Figure 11.8
11.5 Molecular Methods for Mutagenesis
• Synthetic DNA
• Systems are available for de novo
synthesis
of DNA
• Oligonucleotides of 100 bases can be
made
• Multiple oligonucleotides can be ligated
together
• Synthesized DNA is used for primers and
probes, and in site-directed mutagenesis
© 2015 Pearson Education, Inc.
George Church
11.5 Molecular Methods for Mutagenesis
• Site-directed mutagenesis: performed in vitro
and introduces mutations at a precise location
• Can be used to assess the activity of specific amino
acids in a protein
• Structural biologists have gained significant insight
using this tool
© 2015 Pearson Education, Inc.
Michael Smith
Nobel Prize1993
Figure 11.9
11.5 Molecular Methods for Mutagenesis
Cassette mutagenesis and
knockout mutations
• DNA fragment can be cut, excised,
and replaced by a synthetic DNA
fragment (DNA cassettes or
cartridges)
• The process is known as cassette
mutagenesis
• Gene disruption occurs when
cassettes are inserted into the
middle of the gene = knockout
mutations
© 2015 Pearson Education, Inc.
Figure 11.10
11.6 Gene Fusions and Reporter Genes
• Reporter genes
• Encode proteins that are easy to
detect and assay
• Examples: lacZ, luciferase, GFP
genes
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Figure 11.11
11.6 Gene Fusions and Reporter Genes
Gene fusions
Promoters or coding
sequences of genes of
interest can be
swapped with those of
reporter genes to
elucidate gene
regulation under
various conditions
© 2015 Pearson Education, Inc.
Figure 11.12
11.7 Plasmids as Cloning Vectors
• Plasmids are natural vectors and have useful
properties as cloning vectors
• Small size; easy to isolate DNA
• Independent origin of replication
• Multiple copy number; get multiple copies of cloned
gene per cell
• Presence of selectable markers
• Vector transfer is carried out by chemical
transformation or electroporation
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11.7 Plasmids as Cloning Vectors
• pUC19 is a common cloning vector
• Modified ColE1 plasmid
• Contains ampicillin-resistance
• Contains multiple cloning site within lacZ gene
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11.7 Plasmids as Cloning Vectors
© 2015 Pearson Education, Inc.
Figure 11.13
11.7 Plasmids as Cloning Vectors
• Blue/white screening
• Blue colonies do not have
vector with foreign DNA inserted
• White colonies have foreign
DNA inserted
• Insertional inactivation: lacZ
gene is inactivated by
insertion of foreign DNA
• Inactivated lacZ cannot process
Xgal; blue color does not
develop
© 2015 Pearson Education, Inc.
Figure 11.14
11.7 Plasmids as Cloning Vectors
• Other plasmid vectors
• Some vectors developed for cloning DNA products
made by PCR
• Some vectors select for recombinant DNA using viability
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Figure 11.15
11.8 Hosts for Cloning Vectors
• Ideal hosts should be:
• Capable of rapid growth in inexpensive medium
• Nonpathogenic
• Capable of incorporating DNA
• Genetically stable in culture
• Equipped with appropriate enzymes to allow replication
of the vector
• Escherichia coli, Bacillus subtilis, Saccharomyces
cerevisiae, others?
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11.8 Hosts for Cloning Vectors
© 2015 Pearson Education, Inc.
Figure 11.16
11.9 Shuttle Vectors and Expression Vectors
• Shuttle vectors: vectors that are stably maintained
in two or more unrelated host organisms (e.g., E.
coli and B. subtilis or E. coli and yeast)
• Bacterial plasmid engineered to function in
eukaryotes
• Add a eukaryotic origin of replication
• Add a centromere recognition sequence
© 2015 Pearson Education, Inc.
oriC
Ampicillin
resistance
t/pa
ESM
Promoter
E. coli and S. cerevisiae
oriY
t/pa
CEN
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Promoter
MCS
Figure 11.17
11.9 Shuttle Vectors and Expression Vectors
• Expression vectors: allow experimenter to control
the expression of cloned genes
• Based on transcriptional control
• Allow for high levels of protein expression
• Strong promoters
• Efficient operators
• Effective transcription terminators are used to prevent
expression of other genes on the plasmid
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11.9 Shuttle Vectors and Expression Vectors
• In T7 expression vectors, cloned
genes are placed under control of
the T7 promoter = hyperexpression
• Gene for T7 RNA polymerase
present and under control of easily
regulated system (e.g., lac)
• T7 RNA polymerase recognizes only T7 promoters
• Transcribes only cloned genes
• Shuts down host transcription
© 2015 Pearson Education, Inc.
11.9 Shuttle Vectors and Expression Vectors
© 2015 Pearson Education, Inc.
Figure 11.19
11.9 Shuttle Vectors and Expression Vectors
• mRNA produced must be efficiently translated;
problems with this always occur
• Bacterial ribosome-binding sites are not present in
eukaryotic genomes
• Differences in codon usage between organisms
• Eukaryotic genes containing introns will not be
expressed properly in prokaryotes
© 2015 Pearson Education, Inc.
11.10 Other Cloning Vectors
• Bacteriophage lambda
• Modified lambda makes a good cloning vector
• Well-understood biology
• Can hold larger amounts of DNA than most plasmids
• DNA can be efficiently packaged in vitro
• Can efficiently infect suitable host particles
• Lambda vectors are useful in cloning large DNA
fragments
© 2015 Pearson Education, Inc.
11.10 Other Cloning Vectors
Cloning with lambda
1.
Isolate vector DNA from phage
particles, and cut it with the
appropriate restriction enzyme
2.
Connec the lambda fragments
to foreign DNA using DNA
ligase
3.
Package the DNA by adding
cell extracts containing the
head and tail proteins
4.
Infect E. coli cells, and isolate
phage clones by picking
plaques
5.
Check the recombinant phage
for the presence of foreign
DNA
© 2015 Pearson Education, Inc.
Figure 11.20a
11.10 Other Cloning Vectors
• Cosmids: plasmid vectors containing the cos site
from the lambda genome
• Can be packaged into lambda virions
• Inserts as large as 50 kilobases are accepted
• Phage particles are more stable than plasmids
• Specialized vectors for genome analysis exist
• Bacterial artificial chromosomes (BACs): Constructed
from the F plasmid
• Yeast artificial chromosomes (YACs)
© 2015 Pearson Education, Inc.
11.10 Vectors for Genomic Cloning and
Sequencing
• Yeast artificial chromosomes (YACs)
• Can accommodate 200–800 kilobases of cloned DNA
• Replicate like normal yeast chromosomes
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Figure 11.22
11.11 Expressing Mammalian Genes in
Bacteria
• Biotechnology
• Use of living organisms for industrial or commercial
applications
• Genetically modified organism (GMO)
• Organism whose genome has been altered
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11.12 Somatotropin and Other Mammalian
Proteins
• Insulin was the first human
protein made commercially by
genetic engineering
• Somatotropin, a growth hormone, is another widely
produced hormone
• Cloned as cDNA from the mRNA
• Recombinant bovine somatotropin (rBST) is commonly
used in the dairy industry; stimulates milk production in
cows
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11.12 Somatotropin and Other Mammalian
Proteins
© 2015 Pearson Education, Inc.
Figure 11.26
11.13 Transgenic Organisms in Agriculture and
Aquaculture
• Transgenic organism
• Organism that contains a gene from another organism
• Plants can be genetically modified through several
approaches, including:
• Electroporation, Particle gun methods, plasmids from
bacterium Agrobacterium tumefaciens
• Many successes in plant genetic engineering;
several transgenic plants are in agricultural
production
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11.13 Transgenic Organisms in Agriculture and
Aquaculture
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Figure 11.28
11.13 Transgenic Organisms in Agriculture and
Aquaculture
• The plant pathogen Agrobacterium tumefaciens
can be used to introduce DNA into plants
• A. tumefaciens contains the Ti plasmid, which is
responsible for virulence
• The Ti plasmid contains genes that mobilize DNA
for transfer to the plant
• The segment of the Ti plasmid that is transferred to the
plant is called the T-DNA
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11.13 Transgenic Organisms in Agriculture and
Aquaculture
Kanamycin
resistance
Mobilized region
Foreign
DNA
1. Transfer to
E.coli cells.
"Disarmed"
Ti plasmid
Origin
A. tumefaciens
2. Transfer by
conjugation.
Spectinomycin
resistance
Origin E. coli
Cloning vector
3. Transfer to
plant cells.
4. Grow transgenic
plants from
plant cells.
Chromosomes
D-Ti
E. coli
Nucleus
A. tumefaciens
Plant cell
© 2015 Pearson Education, Inc.
Figure 11.27
11.13 Transgenic Organisms in Agriculture and
Aquaculture
• Several areas are targeted for genetic
improvements in plants, including resistance to
herbicides, insects, and microbial disease, as well
as improved product quality
• Plants are engineered to have herbicide
resistance to protect them from herbicides applied
to kill weeds (e.g. glyphosate)
© 2015 Pearson Education, Inc.
11.13 Transgenic Organisms in Agriculture and
Aquaculture
Glyphosate Sensitive
Glyphosate Resistant
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Figure 11.29
11.13 Transgenic Organisms in Agriculture and
Aquaculture
• One widely used approach for genetically engineering insect
resistance in plants involves introducing genes encoding the
toxic protein of Bacillus thuringiensis (Bt toxin)
Beet army worm infestation of tobacco
-Bt
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+Bt
Figure 11.30
11.13 Transgenic Organisms in Agriculture and
Aquaculture
• Genetic engineering can be used to develop
transgenic animals
• Transgenic animals are useful for:
• Improving livestock and other animals for human
consumption
© 2015 Pearson Education, Inc.
Figure 11.31
11.14 Genetically Engineered Vaccines
Vector vaccine
Vaccine made by inserting
genes from a pathogenic virus
into a relatively harmless carrier
virus (e.g., vaccinia virus)
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Figure 11.32
11.14 Genetically Engineered Vaccines
• Subunit vaccines
• Contain only a specific protein or proteins from a
pathogenic organism (e.g., coat protein of a virus)
• Preparation of a viral subunit vaccine
• Fragmentation of viral DNA by restriction enzymes
• Cloning of viral coat protein genes into a suitable vector
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11.15 Mining Genomes
• Gene mining
• The process of isolating potentially useful novel genes
from the environment without culturing the organism
© 2015 Pearson Education, Inc.
Figure 11.33
11.16 Engineering Metabolic Pathways
• Pathway engineering
• The process of assembling a new or improved
biochemical pathway using genes from one or more
organisms
© 2015 Pearson Education, Inc.
Figure 11.34
NEST: Novel Enteric Synbiotic Technology
• Probiotic = live microorganism that provides a health
benefit
• Prebiotic = substances that induce the growth or activity of
microorganisms
• Synbiotic = Combination of probiotic and prebiotic that
provides a synergistic benefit
© 2015 Pearson Education, Inc.
NEST: Novel Enteric Synbiotic Technology
1
2
3
1
2
3
• Our goal is to engineer selective metabolism of a rare carbohydrate
into a probiotic
• In this system – the addition of a selective prebiotic will regulate the
proliferation and persistence of the engineered microbe
• This microbe can then be used to deliver beneficial proteins to the
intestine
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Synbiotic Engineering
‘Common’
Nutrient
POSITIVE SELECTION
‘Rare’
Nutrient
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11.17 Synthetic Biology
• Synthetic biology – using genetic engineering to
create novel biological systems out of available
parts (biobricks)
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11.17 Synthetic Biology
• Synthetic biology – using genetic engineering to
create novel biological systems out of available
parts (biobricks)
© 2015 Pearson Education, Inc.
Figure 11.35