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Cloning Promoters
Kelli Henry
April 27, 2009
Where in the Seed Is Your Gene of Interest Transcribed?
At What Stage of Development?
Seed Coat
Endosperm
Embryo
Zygote
Preglobular
Heart
Linear Cotyledon
Globular
Torpedo
Bending Cotyledon
Transition
Linear Cotyledon
Mature Green
What Processes Occur During Seed Development?
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How Do Genes Get Expressed in a Eukaryote?
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Leaf
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Seed
QuickT ime ™an d a
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Do Arabidopsis leaves
and seeds have the same
genes?
Do they express the same
genes?
Common Reporter Genes in Plants
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The jellyfish green fluorescent
protein (GFP) fluoresces green
under UV light
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Endosperm
The E. coli enzyme betaglucuronidase (GUS) converts a
colorless substrate (X-gluc) to a
blue precipitate
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GUS
colorless substrate
------>
blue product
Which is more sensitive?
Whole Seed
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Chalazal Endosperm
Where and When Is Your Gene of Interest Transcribed?
STOP
ATG
STOP
TATA box
3’ UTR
5’ UTR
3’ UTR
Upstream region contains the “ON
switch” for your gene of interest
Take the upstream region from genomic
DNA and fuse to GFP reporter gene
GFP
TATA box
3’ UTR
5’ UTR
Transform into Arabidopsis plant
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
GFP expression indicates where/when your gene of
interest is transcribed
How Can Your Promoter of Interest Be Isolated and
Amplified?
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™
QuickTime™ and
and a
a
TIFF
TIFF (LZW)
(LZW) decompressor
decompressor
are
are needed
needed to
to see
see this
this picture.
picture.
How Can Plant Cells Be Transformed?
What is the
structure of a
plant cell?
Do plant cells
have plasmids?
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How can you take advantage of a natural process to transform a plant?
How to Transform Arabidopsis
Use the natural genetic engineering
ability of Agrobacterium tumefaciens
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Agrobacterium causes crown gall
disease by transferring DNA (TDNA) from part of the
Agrobacterium tumor-inducing (Ti)
plasmid into the plant genome
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Agrobacterium Naturally Transforms Plants
T-DNA Inserts Randomly Into Plant Genome
Agrobacterium Ti-Plasmid
Figure 20-25
~5kb For Your Vector
What Is Totipotency?
How does A. tumefaciens
infect a plant?
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QuickTime™
and
decompressor
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andaa
decompressor
are needed
to see this picture.
decompressor
are
are needed
needed to
to see
seethis
thispicture.
picture.
Where and When Is Your Gene of Interest Transcribed?
STOP
ATG
STOP
TATA box
3’ UTR
5’ UTR
3’ UTR
Upstream region contains the “ON
switch” for your gene of interest
Take the upstream region from genomic
DNA and fuse to GFP reporter gene
GFP
TATA box
3’ UTR
5’ UTR
Transform into Arabidopsis plant
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
GFP expression indicates where/when your gene of
interest is transcribed
How Can You Get the Promoter to Insert into a
Vector in the Correct Orientation?
GFP
TATA box
3’ UTR
5’ UTR
vs
GFP
Use Directional TOPO Cloning
Directional TOPO Cloning
A method to clone your PCR product in a 5’ to 3’ orientation
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Why use a
proofreading
polymerase?
Promoter of Interest
ATG
TAC
Why Use a Proofreading Polymerase?
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iProof Polymerase provides 3’ to 5’ exonuclease activity
unlike Taq polymerases. This proofreading function allows it
to correct nucleotide misincorporation errors for much
higher fidelity of amplification.
Could one mutation could affect the transcription of your gene?
Directional TOPO Cloning
A method to clone your PCR product in a 5’ to 3’ orientation
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decompressor
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Promoter of Interest
ATG
TAC
Directional TOPO Cloning
Topoisomerase I recognition sites
Extra sequence is cleaved
off in E. coli
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What Is Topoisomerase I?
Topoisomerase I relieves supercoils
in circular DNA plasmids by nicking
one of the strands of the DNA
double helix, twisting it around the
other strand, and re-ligating the
nicked strand
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If you wanted topoisomerase to insert a
segment of DNA, what stage of the
reaction would you want the TOPO
vector to be suspended in?
Directional TOPO Cloning
Topoisomerase I recognition sites
Topoisomerase I
1. Cleaves and linearizes the pENTR
vector, allowing insertion of the PCR
fragment
2. Ligates the vector
How Can Your Promoter of Interest Be Isolated and
Amplified?
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
QuickTime™
QuickTime™ and
and a
a
TIFF
TIFF (LZW)
(LZW) decompressor
decompressor
are
are needed
needed to
to see
see this
this picture.
picture.
How Do You Screen for Recombinant Plasmids with the Insert
in the Correct Orientation?
Isolate plasmid DNA from E. coli colonies and do restriction digest
Expected Sizes (bp)
Correct
Orientation
AT3G05860 promoter in
pENTR x SacII x PacI
1435
2745
6108
5090
4072
3054
Incorrect
Orientation
3987
193
One Cut
Vector
Only
4180
2600
2036
1636
Correct orientation
Incorrect orientation
5/5 E. coli colonies contain plasmids with the insert in the correct orientation
Sequence to make sure there are no mutations that could affect transcription
Transfer the Promoter into the T-DNA Shuttle Vector
LB
BastaR
GFP
TATA box
3’ UTR
5’ UTR
GUS
RB
Transfer the Promoter into the T-DNA Shuttle Vector
Use Shuttle vector and Helper Ti plasmid instead of Ti plasmid
Agrobacterium Ti-Plasmid
Figure 20-25
~5kb For Your Vector
How Can the Promoter Be Transferred from pENTR
Vector to the T-DNA Vector?
gene
attB1
ccdB
gene
attL1
attR1
attL2
Entry
Clone
KanR
ccdB
+
attR2
attP1
Donor
Vector
AmpR
KanR
LR Clonase™ II
Expression
Clone
attP2
Destination
Vector
attB2
+
AmpR
90-99% correct clones
on Amp plates
Use homologous recombination
Phage lambda Recombination in E. coli
How Can You Use Agrobacterium to Transfer Your
Plasmid DNA into Arabidopsis?
Transferred DNA (T-DNA) contains genes encoding tumorinducing hormones and opines (a carbon/nitrogen source
that can only be metabolized by Agrobacterium) between
LB and RB
Replace these genes with your promoter, reporter genes
and selectable marker
LB
BastaR
GFP
TATA box
3’ UTR
5’ UTR
GUS
RB
How Agrobacterium Transforms Plants
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Dip Arabidopsis in a Solution of Agrobacterium
Containing Your T-DNA
Floral dipping transforms the female reproductive tissues that give
rise to seeds after fertilization. ~1% of seeds are transformed.
How do you select for transformed seeds?
Select for Transformed Plants
1. Sow T1 seeds and treat with Basta (herbicide) to select for
transformed seeds
–Transformed seeds will express the Basta resistance gene
on the T-DNA vector
2. T1 seeds develop into T1 plants (hemizygous), which
produce T2 seeds (1 WT: 2 hemi: 1 homo)
3. Observe reporter gene expression in T2 seeds at different
stages of development under the microscope
T0 (dipped)
T1 seeds (hemi)
T1 plants (hemi)
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BastaR (green)
BastaS (yellow)
T2 seeds (1:2:1)
Where/When Is Your Gene of Interest Transcribed?
STOP
ATG
STOP
TATA box
3’ UTR
5’ UTR
3’ UTR
Upstream region contains the “ON
switch” for your gene of interest
LB
BastaR
GFP
TATA box
3’ UTR
GUS
RB
5’ UTR
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TIF F ( LZ W) decompressor
are needed to see this pict ur e.
GFP
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TIF F ( LZ W) decompressor
are needed to see this pict ur e.
Endosperm
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GUS
Arabidopsis Silique
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Whole Seed Chalazal Endosperm