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Cloning Promoters. Kelli Henry April 27, 2009. Preglobular. Globular. Zygote. Transition. Heart. Torpedo. Linear Cotyledon. Linear Cotyledon. Bending Cotyledon. Mature Green. Where in the Seed Is Your Gene of Interest Transcribed? At What Stage of Development?. Seed Coat. Endosperm.

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cloning promoters

Cloning Promoters

Kelli Henry

April 27, 2009

slide2

Preglobular

Globular

Zygote

Transition

Heart

Torpedo

Linear Cotyledon

Linear Cotyledon

Bending Cotyledon

Mature Green

Where in the Seed Is Your Gene of Interest Transcribed?At What Stage of Development?

Seed Coat

Endosperm

Embryo

how do genes get expressed in a eukaryote
How Do Genes Get Expressed in a Eukaryote?

Leaf

Seed

Do Arabidopsis leaves and seeds have the same genes?

Do they express the same genes?

common reporter genes in plants
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

Endosperm

Whole Seed

Chalazal Endosperm

Which is more sensitive?

where and when is your gene of interest transcribed

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

GFP expression indicates where/when your gene of interest is transcribed

Where and When Is Your Gene of Interest Transcribed?

STOP

ATG

STOP

TATA box

3’ UTR

5’ UTR

3’ UTR

how can plant cells be transformed
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
How to Transform Arabidopsis

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

slide10

Agrobacterium Naturally Transforms Plants

T-DNA Inserts Randomly Into Plant Genome

figure 20 25
Figure 20-25

Agrobacterium Ti-Plasmid

~5kb For Your Vector

what is totipotency
What Is Totipotency?

How does A. tumefaciens infect a plant?

where and when is your gene of interest transcribed1

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

GFP expression indicates where/when your gene of interest is transcribed

Where and When Is Your Gene of Interest Transcribed?

STOP

ATG

STOP

TATA box

3’ UTR

5’ UTR

3’ UTR

how can you get the promoter to insert into a vector in the correct orientation

TATA box

3’ UTR

5’ UTR

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
Directional TOPO CloningA method to clone your PCR product in a 5’ to 3’ orientation

Why use a proofreading polymerase?

ATG

TAC

Promoter of Interest

why use a proofreading polymerase
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?

directional topo cloning a method to clone your pcr product in a 5 to 3 orientation1
Directional TOPO CloningA method to clone your PCR product in a 5’ to 3’ orientation

ATG

TAC

Promoter of Interest

directional topo cloning
Directional TOPO Cloning

Topoisomerase I recognition sites

Extra sequence is cleaved off in E. coli

what is topoisomerase i
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

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 cloning1
Directional TOPO Cloning

Topoisomerase I recognition sites

Topoisomerase I

Cleaves and linearizes the pENTR vector, allowing insertion of the PCR fragment

2. Ligates the vector

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

Incorrect Orientation

Vector

Only

One Cut

AT3G05860 promoter in pENTR x SacII x PacI

4180

2600

3987

193

1435

2745

6108

5090

4072

3054

2036

1636

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

slide24

Transfer the Promoter into the T-DNA Shuttle Vector

Use Shuttle vector and Helper Ti plasmid instead of Ti plasmid

figure 20 251
Figure 20-25

Agrobacterium Ti-Plasmid

~5kb For Your Vector

how can the promoter be transferred from pentr vector to the t dna vector

90-99% correct clones

on Amp plates

How Can the Promoter Be Transferred from pENTR Vector to the T-DNA Vector?

+

+

LR Clonase™ II

Use homologous recombination

how can you use agrobacterium to transfer your plasmid dna into arabidopsis

Replace these genes with your promoter, reporter genes and selectable marker

3’ UTR

5’ UTR

LB

BastaR

GFP

GUS

RB

TATA box

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

dip arabidopsis in a solution of agrobacterium containing your t dna
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)

T0 (dipped)

T1 seeds (hemi)

BastaR (green)

T1 plants (hemi)

BastaS (yellow)

T2 seeds (1:2:1)

Select for Transformed Plants

3. Observe reporter gene expression in T2 seeds at different stages of development under the microscope

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

Endosperm

GFP

3’ UTR

5’ UTR

Whole Seed

Chalazal Endosperm

GUS

LB

BastaR

GFP

GUS

RB

TATA box

Arabidopsis Silique

Where/When Is Your Gene of Interest Transcribed?
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