Cloning Promoters
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Cloning Promoters
Kelli Henry
April 27, 2009
Mature Green
Zygote Preglobular Globular Transition
Heart Torpedo Linear Cotyledon
Bending CotyledonLinear Cotyledon
Where in the Seed Is Your Gene of Interest Transcribed?At What Stage of Development?
Seed Coat
Endosperm
Embryo
What Processes Occur During Seed Development?
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How Do Genes Get Expressed in a Eukaryote?
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Do Arabidopsis leaves and seeds have the same
genes?
Do they express the same genes?
Common Reporter Genes in Plants
The jellyfish green fluorescent protein (GFP) fluoresces green under UV light
The E. coli enzyme beta-glucuronidase (GUS) converts a colorless substrate (X-gluc) to a blue precipitate
GUS colorless substrate ------> blue
product
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Endosperm
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Which is more sensitive?
Where and When Is Your Gene of Interest Transcribed?
TATA box
ATG STOP
5’ UTR 3’ UTR
STOP
3’ UTR
Upstream region contains the “ON switch” for your gene of interest
TATA box GFP5’ UTR
Take the upstream region from genomic DNA and fuse to GFP reporter gene
Transform into Arabidopsis plant
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GFP expression indicates where/when your gene of
interest is transcribed
3’ UTR
How Can Your Promoter of Interest Be Isolated and Amplified?
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How Can Plant Cells Be Transformed?
What is the structure of a
plant cell?
Do plant cells have plasmids?
How can you take advantage of a natural process to transform a plant?
How to Transform Arabidopsis
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Use the natural genetic engineering ability of Agrobacterium tumefaciens
Agrobacterium causes crown gall disease by transferring DNA (T-DNA) 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
Figure 20-25
Agrobacterium Ti-Plasmid
~5kb For Your Vector
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What Is Totipotency?
How does A. tumefaciens infect a plant?
Where and When Is Your Gene of Interest Transcribed?
TATA box
ATG STOP
5’ UTR 3’ UTR
STOP
3’ UTR
Upstream region contains the “ON switch” for your gene of interest
TATA box GFP5’ UTR
Take the upstream region from genomic DNA and fuse to GFP reporter gene
Transform into Arabidopsis plant
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
GFP expression indicates where/when your gene of
interest is transcribed
3’ UTR
How Can You Get the Promoter to Insert into a Vector in the Correct Orientation?
TATA box GFP5’ UTR3’ UTR
GFP
TATA box
5’ UTR 3’ UTR
Use Directional TOPO Cloning
vs
Directional TOPO Cloning A method to clone your PCR product in a 5’ to 3’ orientation
ATG
TACPromoter of Interest
Why use a proofreading polymerase?
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Why Use a Proofreading Polymerase?
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?
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Directional TOPO Cloning A method to clone your PCR product in a 5’ to 3’ orientation
ATG
TACPromoter of Interest
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Directional TOPO Cloning
Extra sequence is cleaved off in E. coli
Topoisomerase I recognition sites
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What Is Topoisomerase I?
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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
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?
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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?
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How Do You Screen for Recombinant Plasmids with the Insert in the Correct Orientation?
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
1636
2036
3054407250906108
AT3G05860 promoter in pENTR x SacII x PacI 1435
2745
Expected Sizes (bp)
Correct Orientation
Incorrect Orientation One Cut
41803987193
Vector Only
2600
Isolate plasmid DNA from E. coli colonies and do restriction digest
Transfer the Promoter into the T-DNA Shuttle Vector
5’ UTR3’ UTR
GUSGFPTATA box RBLB BastaR
Transfer the Promoter into the T-DNA Shuttle Vector
Use Shuttle vector and Helper Ti plasmid instead of Ti plasmid
Figure 20-25
Agrobacterium Ti-Plasmid
~5kb For Your Vector
ExpressionClone
gene
attB1 attB2
AmpR
attP1 attP2
ccdB
DonorVector
KanR
attR1 attR2
ccdB
DestinationVector
AmpR
gene
attL1 attL2
EntryClone
KanR
90-99% correct cloneson Amp plates
How Can the Promoter Be Transferred from pENTR Vector to the T-DNA Vector?
+ +
LR Clonase™ II
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 tumor-inducing hormones and opines (a carbon/nitrogen source that can only be metabolized by Agrobacterium) between LB and RB
5’ UTR3’ UTR
GUSGFPTATA box RBLB BastaR
Replace these genes with your promoter, reporter genes and selectable marker
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
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BastaS (yellow)
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
3. Observe reporter gene expression in T2 seeds at different stages of development under the microscope
2. T1 seeds develop into T1 plants (hemizygous), which produce T2 seeds (1 WT: 2 hemi: 1 homo)
T0 (dipped)
T1 seeds (hemi)
T1 plants (hemi)
T2 seeds (1:2:1)
Where/When Is Your Gene of Interest Transcribed?
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Endosperm
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Whole Seed Chalazal Endosperm
GUS
GFP
Upstream region contains the “ON switch” for your gene of interest
TATA box
ATG STOP
5’ UTR 3’ UTR
STOP
3’ UTR
5’ UTR3’ UTR
GUSGFPTATA box RBLB BastaR
Arabidopsis Silique
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