Download E. coli - Sonoma Valley High School

Chapter 17
Lecture Outline
See separate PowerPoint slides for all figures and
tables pre-inserted into PowerPoint without notes and
animations.
1
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Biotechnology
Chapter 17
2
Recombinant DNA
• Restriction endonucleases revolutionized
molecular biology
• Enzymes that cleave DNA at specific sites
– Used by bacteria against viruses
• Restriction enzymes significant
– Allow a form of physical mapping that was
previously impossible
– Allow the creation of recombinant DNA
molecules (from two different sources)
3
3 Types of Restriction Enzymes
•Type I and III cleave with less precision and
are not used in manipulating DNA
•Type II
– Recognize specific DNA sequences
– Cleave at specific site within sequence
– Can lead to “sticky ends” that can be joined
• Blunt ends can also be joined
4
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
EcoRI
DNA
duplex
Restriction sites
EcoRI
G
A
A
T
T
C
G
A
A
T
T
C
C
T
T
A
A
G
C
T
T
A
A
G
EcoRI
Restriction endonuclease
cleaves the DNA
A
A
T
T
EcoRI
Restriction endonuclease
cleaves the DNA
C
G
G
C
Sticky ends
T
A
T
T
A
A
Sticky ends
G
C
A
T
A
A
T
T
C
G
DNA from another source cut with the
same restriction endonuclease is added.
A
A
T
T
C
G
G
C
A
T
A
T
T
A
T
A
C
G
Recombinant DNA molecule
DNA ligase
joins the strands.
5
DNA ligase
– Joins the two fragments forming a stable DNA
molecule
– Catalyzes formation of a phosphodiester bond
between adjacent phosphate and hydroxyl
groups of DNA nucleotides
– Same enzyme joins Okazaki fragments on
lagging strand in replication
6
Gel Electrophoresis
•
•
•
•
•
Separate DNA fragments by size
Gel made of agarose or polyacrylamide
Submersed in buffer that can carry current
Subjected to an electrical field
Negatively-charged DNA migrates towards the
positive pole
• Larger fragments move slower, smaller move
faster
• DNA is visualized using fluorescent dyes
7
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Restriction Enzyme Digestion
Gel Electrophoresis
DNA samples are cut with restriction enzymes in three
different reactions producing different patterns off ragments.
Samples from the restriction enzyme digests are introduced into the gel.
Electric current is applied causing fragments to migrate through the gel.
Restriction endonuclease
1 cut site
Reaction Reaction Reaction
1
2
3
Power
source
Reaction 1
Short segment
Long segment
Mixture of DNA
fragments of
different sizes in
solution placed at
the top of “lanes” in
the gel
Lane
Restriction endonuclease
2 cut site
–
Cathode
Reaction 2
Gel
Medium segment
Medium segment
Restriction
endonuclease 3
+
Reaction 3
Anode
Buffer
Long segment
a.
Short segment
b.
8
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Visualizing Stained Gel
Electrophoresis in the Laboratory
Gel is stained with a dye to allow
the fragments to be visualized.
Longer
fragments
Shorter
fragments
c.
d.
d: Courtesy of Biorad Laboratories
9
Transformation
• Introduction of foreign DNA from an
outside source into a cell
• Natural process in many species
– E. coli does not
• Temperature shifts can induce artificial
transformation in E. coli
• Transgenic organisms are all or part
transformed cells
10
Molecular Cloning
• Clone – genetically identical copy
• Molecular cloning – isolation of a specific
DNA sequence (usually protein-encoding)
– Sometimes called gene cloning
• The most flexible and common host for
cloning is E. coli
– Vector – carries DNA in host and can
replicate in the host
– Each host–vector system has particular uses
11
Vectors
• Plasmids
– Small, circular chromosomes
– Used for cloning small pieces of DNA
– 3 components
• Origin of replication – allows independent
replication
• Selectable marker – allows presence of plasmid to
be easily identified
• Multiple cloning site (MCS)
12
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
A Plasmid Vector
Restriction
endonuclease
Foreign
DNA
lacZ gene
Transform
No DNA
inserted
Medium contains
ampicillin and X-gal
Ampicillin
resistance
gene
Restriction enzymes
cuts within
the laxZ gene
Foreign DNA
and DNA ligase
are added
DNA
inserted
Active lacZ
gene produces
blue colonies
Inactive lacZ
gene produces
white colonies
Transform
13
• Artificial chromosomes
– Plasmids have limited insert size
– Yeast artificial chromosomes (YACs)
– Bacterial artificial chromosomes (BACs)
– Allow for larger insert for large-scale analysis
of genomes
14
Plant genetic engineering
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Gene of
interest
Plasmid
Agrobacterium
Plant nucleus
1. Plasmid is
removed and cut open
with restriction
endonuclease.
2. A gene of interest is
isolated from the DNA
of another organism
and inserted into the
plasmid. The plasmid
is put back into the
Agrobacterium.
3. When used to infect plant cells,
Agrobacterium duplicates part
of the plasmid and transfers the
new gene into a chromosome of
the plant cell.
4. The plant cell divides, and each
daughter cell receives the new
gene. These cultured cells can
be used to grow a new plant
with the introduced gene.
15
Polymerase chain reaction (PCR)
• Developed by Kary Mullis
– Awarded Nobel Prize
• Allows the amplification of a small DNA fragment
using primers that flank the region
• Each PCR cycle involves three steps:
1. Denaturation (high temperature)
2. Annealing of primers (low temperature)
3. DNA synthesis (intermediate temperature)
• Taq polymerase
16
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA segment
to be amplified
5´
3´
3´
5´
PCR
machine
1. Sample is first heated
to denature DNA.
DNA is denatured
into single strands
5´
3´
3´
5´
2. DNA is cooled to a
lower temperature
to allow annealing
of primers.
5´
3´
Primers anneal to DNA
3´
5´
17
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
After 20 cycles, a
single fragment
produces over one
million (220) copies!
3. DNA is heated to
72°C, the optimal
temperature for Taq
DNA polymerase to
extend primers
5´
3´
3´
5´
Taq DNA polymerase
3´
5´
3´
5´
3´
3´
5´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
3´
5´
3´
5´
5´
5´
3´
Cycle 2:
4 copies
Cycle 3:
8 copies
5´
3´
3´
5´
3´
5´
3´
5´
3´
3´
5´
5´
3´
5´
5´
3´
3´
5´
3´
5´
5´
3´
3´
5´
18
• Applications of PCR
– Allows the investigation of minute samples of
DNA
– Forensics – drop of blood, cells at base of a
hair
– Detection of genetic defects in embryos by
analyzing a single cell
– Analysis of mitochondrial DNA from early
human species
19
DNA Libraries
• A collection of DNAs in a vector that taken
together represent the complex mixture of
DNA
• Genomic library – representation of the
entire genome in a vector
– Genome is randomly fragmented
– Inserted into a vector
– Introduced into host cells
– Usually constructed in BACs
20
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Plasmid Library
DNA fragments
from source DNA
DNA inserted
into plasmid vector
Transformation
Each cell contains a
single fragment. All cells
together are the library.
21
Complementary DNA (cDNA)
– DNA copies of mRNA
– mRNA isolated
• Represents only actively used genes
• No introns
– Use reverse transcriptase to make cDNA
– cDNA used to make library
– All genomic libraries from a cell will be the
same but cDNA libraries can be different
22
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
exons
introns
1
1
2
2
3
3
4
4
Eukaryotic DNA template
Transcription
5´ cap
3´ poly- A tail
Primary RNA transcript
Introns are cut out,
and coding regions are
spliced together.
3´ poly- A tail
5´ cap
Mature RNA transcript
Isolation of mRNA
Addition of reverse
transcriptase
23
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Reverse
transcriptase
Reverse
transcriptase
utilizes mRNA
to create cDNA.
Addition of mRNAdegrading enzymes
mRNA–cDNA hybrid
Degraded
mRNA
DNA polymerase
Double-stranded cDNA
with no introns
24
Analyzing and Creating DNA
Differences
• Molecular hybridization
– Technique used to identify specific DNAs in
complex mixtures such as libraries
– Also termed annealing
– Known single-stranded DNA or RNA is
labeled
– Used as a probe to identify its
complement via specific base-pairing
25
• Molecular hybridization is the most
common way of identifying a clone in a
DNA library
• This process involves three steps:
1. Plating the library
•
Physically the library is a collection of bacteria or
viruses in bacteria
2. Replicating the library
3. Screening the library
•
Probe is specific sequence of interest
26
• Southern blotting
– Sample DNA is digested by restriction
enzymes and separated by gel
electrophoresis
– Double-stranded DNA denatured into singlestrands
– Gel “blotted” with filter paper to transfer DNA
– Filter is incubated with a labeled probe
consisting of purified, single-stranded DNA
corresponding to a specific gene
27
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
1. DNA in the gel is
transferred, or
“blotted, ”onto the
nitrocellulose.
Gel
Nitrocellulose
paper now
contains nucleic
acid “print”
Radioactive
probe (singlestranded DNA)
2. Nitrocellulose with
bound DNA is
incubated with
radio actively labeled
nucleic acids and is
then rinsed.
Sealed
container
—AATGG—
—TTACC—
DNA fragments
within bands
3. Photographic film is
laid over the filter
and is exposed only
in areas that contain
radioactivity
(autoradiography).
Bands on the film
represent DNA in the
gel that is
complementary
to the probe
sequence.
Film
Hybridized nucleic acids
Size markers
© SSPL/The Image Works
28
• Northern blotting
– mRNA is separated by electrophoresis and
then blotted onto the filter
• Western blotting
– Proteins are separated by electrophoresis and
then blotted onto the filter
– Detection requires an antibody that can bind
to one protein
29
DNA Fingerprinting
• RFLP analysis
– Restriction fragment length polymorphisms
– Generated by point mutations or sequence
duplications
– Restriction enzyme fragments are often not
identical in different individuals
– Can be detected by Southern blotting
30
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Original Sequence
of Restriction Sites
(no mutations)
Point Mutations
Change the
Sequence of
Restriction Sites
Sequence
Repetitions Can
Occur Between
Restriction Sites
Larger
fragments
restriction endonuclease
cutting sites
+
Single base-pair
change
Smaller
fragments
–
+
–
+
–
+
Sequence duplication
+
a. Three different
DNA duplexes
b. Cut DNA
c. Gel electrophoresis of
restriction fragments
31
• DNA fingerprinting
– Identification technique used to detect
differences in the DNA of individuals
– Short tandem repeats (STRs)
• Typically 2–4 nt long
• Not part of coding or regulatory regions
– Population is polymorphic for these markers
– Using several probes, probability of identity
can be calculated or identity can be ruled out
– Also used to identify remains
32
STR analysis
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
STRs and DNA fingerprinting
1 Control Ladder
2
3
4
5
6
7
bp
DYS19
STR
Y chromosome
202 absent
198
194
190
186
178 absent
172
D12S66
STR
Chromosome 12
168
160
156
152 absent
148 absent
Data provided by Dr. L. Roewer, DNA Laboratory of the Institute for Forensic Medicine of Charité, Berlin
33
“Knockout” mice
• Cloned gene interrupted by replacement with a
marker gene
• Marker gene codes for resistance to the
antibiotic neomycin
• Interrupted gene is introduced into embryonic
stem cells (ES cells)
• ES cells injected into embryo early in
development
34
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
neo
neo
Gene to be knocked out
neo
1. Using recombinant DN A techniques, the gene
encoding resistance to neomycin (neo) is inserted
into the gene of interest, disrupting it. The neo gene
also confers resistance to the drug G418, which kills
mouse cells. This construct is then introduced into
ES cells.
2. In some ES cells, the construct will recombine
with the chromosomal copy of the gene to be
knocked out. This replaces the chromosomal
copy with the neo disrupted construct. This is
the equivalent to a double crossover event in
a genetic cross.
35
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Embryonic stem (ES) cells with
knocked out gene
ES cells
containing
neo
G418-containing
medium
Surrogate mouse
Blastocyst
Dead cells without
knocked out gene
3. The ES cells are placed on G418containing medium. The G418 selects
cells that have had a replacement event,
and now contain a copy of the knocked
out gene.
4. The ES cells containing the knocked out
gene are injected into a blastocyst stage
embryo and then implanted into a female
to complete development.
36
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Heterozygous
mouse carrying
the knockout gene
Homozygous
mouse for the
knockout gene
5. Offspring will contain one chromosome with
the gene of interest knocked out. Genetic
crosses can then produce mice homozygous
for the knocked out gene to assess the
phenotype. This can range from lethality to
no visible effect depending on the gene.
37
RNA interference
• RNAi is another approach to knockdown or
knockout expression of a gene
–
–
–
–
Permanently alters the DNA
Targets a specific RNA sequence to degrade
Not translated into protein
Used to determine gene function in many organisms
38
Medical Applications
• Medically important proteins can be
produced in bacteria
– Human insulin
– Vaccines
– Problem has been purification of desired
proteins from other bacterial proteins
39
Genetically engineering E. coli to make
human insulin
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
In Humans
Promoter
Exon
Intron
In Bacterial Culture
Exon
Intron
Exon
AmpR
β-gal
Bacterial
promoter
Bacterial
promoter
Insulin A chain minus introns
and other “extra” sequence
Transcription
Exon
β-gal
Insulin B chain minus introns
and other “extra” sequence
Exon
Transform into E.coli
Translation
108 amino acids
Preproinsulin
Culture cells
Posttranslational modification
Cut
Purify β-gal-insulin
fusion proteins
Disulfide
bonds form
Cut
A chain
Purify A and Bchains
B chain
Cut
Achain
NH2
Bchain
NH2
Disulfide bond
Disulfide bond
a.
b.
COOH
COOH
Active
insulin
40
• Vaccines
– Subunit vaccines
• Genes encoding a part of the protein coat are
spliced into a fragment of the vaccinia (cowpox)
genome
• Injection of harmless recombinant virus leads to
immunity
– DNA vaccines
• Depend on the cellular immune response (not
antibodies)
41
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
2. Herpes simplex
gene is isolated.
1. DNA is extracted.
3. Vaccinia DNA
is extracted and
cleaved.
Herpes simplex virus
Human immune
response
6. Antibodies directed
against herpes simplex
viral coat are made.
Gene specifying herpes
simplex surface protein
Harmless vaccinia
(cowpox) virus
4. Fragment containing
surface gene combines
with cleaved vaccinia DNA.
5. Harmless engineered
virus (the vaccine) with
surface like herpes
simplex is injected into
the human body .
42
• Gene therapy
– Adding a functional copy of a gene to correct
a hereditary disorder
– Severe combined immunodeficiency disease
(SCID) illustrates both the potential and the
problems
• On the positive side, 15 children treated
successfully are still alive
• On the negative side, three other children treated
have developed leukemia (due to therapy)
43
44
Agricultural Applications
• Ti (tumor-inducing) plasmid
– Most used vector for plant genetic
engineering
– Obtained from Agrobacterium tumefaciens,
which normally infects broadleaf plants
– Part of the Ti plasmid integrates into the plant
DNA and other genes can be attached to it
– However, bacterium does not infect cereals
such as corn, rice, and wheat
45
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
SCIENTIFIC THINKING
Hypothesis: Petunias can acquire tolerance to the herbicide glyphosate by overexpressing EPSP synthase
Prediction: Transgenic petunia plants with a chimeric EPSP synthase gene with strong promoter will be glyphosate tolerant
Test:
1. Use restriction enzymes and ligase to “paste” the cauliflower mosaic virus promoter (35S) to the EPSP synthase gene and insert the
construct in Ti plasmids.
2. Transform Agrobacterium with the recombinant plasmid.
3. Infect petunia cells and regenerate plants. Regenerate uninfected plants as controls.
4. Challenge plants with glyphosate.
35S
EPSP
synthase
Agrobacterium
Glyphosate
Transformed,
regenerated
petunia plant
Ti plasmid
Cultured petunia cells
Non-tolerant
petunia
Tolerant
petunia
Result: Glyphosate kills control plants, but not transgenic plants.
Conclusion: Additional EPSP synthase provides glyphosate tolerance.
FurtherExperiments: The transgenic plants are tolerant, but not resistant (note bleaching at shoot tip). How could you determine if additional
copies of the gene would increase tolerance? Can you think of any downsides to expressing too much EPSP synthase in petunia?
© Rob Horsch, Monsanto Company
46
• Other methods of gene insertion
– Gene guns
• Uses bombardment with tiny gold particles coated
with DNA
• Possible for any species
• Copy number of inserted genes cannot be
controlled
– Modification of Agrobacterium system
– Use of other bacteria like Agrobacterium
47
• Herbicide resistance
– Broadleaf plants have been engineered to be
resistant to the herbicide glyphosate
– Benefits
• Crop resistant to glyphosate would not have to be
weeded
• Single herbicide instead of many types
• Glyphosate breaks down in environment
– In the United States, 90% of soy currently
grown is GM soy
48
• Bt crops
– Insecticidal proteins have been transferred
into crop plants to make them pest-resistant
– Bt toxin from Bacillus thuringiensis
– Use of Bt maize is the second most common
GM crop globally
• Stacked crops
– Both glyphosate-resistant and Bt-producing
49
• Golden rice
– Rice that has been genetically modified to
produce b-carotene (provitamin A)
– Converted in the body to vitamin A
– Interesting for 2 reasons
• Introduces a new biochemical pathway in tissue of
the transgenic plants
• Could not have been done by conventional
breeding as no rice cultivar known produces these
enzymes in endosperm
– Available free with no commercial
entanglements
50
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Daffodil
phytoene
synthase
gene (psy )
Bacterial
carotene
desaturase
gene (crtI )
Daffodil
lycopene
β-cyclase
gene (lcy )
Genes introduced
Into rice genome
Rice
chromosome
psy
crtI
lcy
Expression
In endosperm
Phytoene
synthase
GGPP
β-Cyclase
Carotene
desaturase
Phytoene
Lycopene
β-Carotene
(Provitamin A)
51
Marker Assisted Breeding (MAB)
•
•
•
•
•
Combines classic plant breeding with molecular biology
DNA extracted from leaf tissue of young seedlings
Screen for agriculturally important traits
Screening uses DNA fingerprinting
Seedlings with desired traits raised to maturity
52
• Adoption of genetically modified (GM)
crops has been resisted in some areas
because of questions
– Crop safety for human consumption
– Movement of genes into wild relatives
• No evidence so far but it is not impossible
53
• Biopharming
– Transgenic plants are used to produce
pharmaceuticals
– 1990 – Human serum albumin produced in
genetically engineered tobacco and potato
plants
– In development
• Recombinant subunit vaccines against Norwalk
and rabies viruses
• Recombinant monoclonal antibodies against tooth
decay-causing bacteria
54
• Transgenic animal technology has not
been as successful as that in plants
• Molecular techniques combined with the
ability to clone domestic animals could
produce improved animals for
economically desirable traits
• Main use thus far has been engineering
animals to produce pharmaceuticals in
milk (also biopharming)
55
Transgenic animals
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Transgenic salmon
Wild salmon
6000
Weight (g)
5000
4000
3000
2000
1000
0
0
100
200 300
400 500
600 700
800 900
Days (from first feeding)
a.
b.
b: © Barrett & MacKay Photo
56