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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