1. Cloning Promoters
Kelli Henry
April 27, 2009

2. Where in the Seed Is Your Gene of Interest Transcribed
Where in the Seed Is Your Gene of Interest Transcribed? At What Stage of Development?
Mature Green
Zygote
Preglobular
Globular
Transition
Heart
Torpedo
Linear Cotyledon
Bending Cotyledon
Seed Coat
Endosperm
Embryo

3. What Processes Occur During Seed Development?

4. 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?

5. 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?

6. 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
TATA box
GFP
5’ UTR
Take the upstream region from genomic DNA and fuse to GFP reporter gene
Transform into Arabidopsis plant
GFP expression indicates where/when your gene of interest is transcribed
3’ UTR

7. How Can Your Promoter of Interest Be Isolated and Amplified?

8. 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?

9. 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

10. Agrobacterium Naturally Transforms Plants
19.10a The Ti plasmid can be used to transfer genes into plants. Flanking sequences TL and TR are required for the transfer of the DNA segment from bacteria to the plant cell.
T-DNA Inserts Randomly Into Plant Genome

11. Agrobacterium Ti-Plasmid
Figure 20-25
~5kb For Your Vector

12. How does A. tumefaciens infect a plant?
What Is Totipotency?
How does A. tumefaciens infect a plant?

13. 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
TATA box
GFP
5’ UTR
Take the upstream region from genomic DNA and fuse to GFP reporter gene
Transform into Arabidopsis plant
GFP expression indicates where/when your gene of interest is transcribed
3’ UTR

14. How Can You Get the Promoter to Insert into a Vector in the Correct Orientation?
GFP
TATA box
3’ UTR
5’ UTR vs
TATA box
5’ UTR
3’ UTR
GFP
Use Directional TOPO Cloning

15. Directional TOPO Cloning A method to clone your PCR product in a 5’ to 3’ orientation
Why use a proofreading polymerase?
ATG
TAC
Promoter of Interest

16. 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?

17. Directional TOPO Cloning A method to clone your PCR product in a 5’ to 3’ orientation
ATG
TAC
Promoter of Interest

18. Directional TOPO Cloning
Topoisomerase I recognition sites
Extra sequence is cleaved off in E. coli
Directional joining of double-stranded DNA using TOPO®-charged oligonucleotides occurs by adding a 3 single-stranded end (overhang) to the incoming DNA. This single-stranded overhang is identical to the 5 end of the TOPO®-charged DNA fragment. At Invitrogen, this idea has been modified by adding a 4 nucleotide overhang sequence to the TOPO®-charged DNA.
In this system, PCR products (up to 5 kb) are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning vector (GTGG) invades the 5 end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. Inserts can be cloned in the correct orientation with high efficiency.

19. 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?

20. Directional TOPO Cloning
Topoisomerase I recognition sites
Topoisomerase I
Cleaves and linearizes the pENTR vector, allowing insertion of the PCR fragment
Directional joining of double-stranded DNA using TOPO®-charged oligonucleotides occurs by adding a 3 single-stranded end (overhang) to the incoming DNA. This single-stranded overhang is identical to the 5 end of the TOPO®-charged DNA fragment. At Invitrogen, this idea has been modified by adding a 4 nucleotide overhang sequence to the TOPO®-charged DNA.
In this system, PCR products (up to 5 kb) are directionally cloned by adding four bases to the forward primer (CACC). The overhang in the cloning vector (GTGG) invades the 5 end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation. Inserts can be cloned in the correct orientation with high efficiency.
2. Ligates the vector

21. How Can Your Promoter of Interest Be Isolated and Amplified?

22. Incorrect Orientation
How Do You Screen for Recombinant Plasmids with the Insert in the Correct Orientation?
1636
2036
3054
4072
5090
6108
AT3G05860 promoter in pENTR x SacII x PacI
1435
2745
Expected Sizes (bp)
Correct Orientation
Incorrect Orientation
One Cut
4180
3987
193
Vector
Only
2600
Isolate plasmid DNA from E. coli colonies and do restriction digest
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

23. Transfer the Promoter into the T-DNA Shuttle Vector
5’ UTR
3’ UTR
GUS
GFP
TATA box RB LB
BastaR

24. Transfer the Promoter into the T-DNA Shuttle Vector
Use Shuttle vector and Helper Ti plasmid instead of Ti plasmid

25. Agrobacterium Ti-Plasmid
Figure 20-25
~5kb For Your Vector

26. How Can the Promoter Be Transferred from pENTR Vector to the T-DNA Vector?+ +
LR Clonase™ II
90-99% correct clones
on Amp plates
In a similar fashion, the expression clone is produced by recombination of the entry clone with a specific destination vector that has the attR1 and attR2 sequences and the same counterselectable marker ccdB. Upon addition of LR Clonase™ the expression clone is produced along with a by-product plasmid containing ccdB. Due to the presence of the ampicillin resistance gene in the destination vector, expression clones are selected on plates containing ampicillin.
Invitrogen offers a wide variety of pre-made destination vectors for expression and functional analysis of your gene of interest in different systems. Most of the destination vectors contain a promoter to drive expression in the host of choice. For example, pET destination vectors offer a T7/lacUV5 promoter for high-level expression in E. coli. pcDNA-DEST vectors provide the CMV promoter for high-level, constitutive expression in mammalian systems such as COS or HEK293 cells.
Use homologous recombination

27. Phage lambda Recombination in E. coli

28. 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’ UTR
3’ UTR
GUS
GFP
TATA box RB LB
BastaR
Replace these genes with your promoter, reporter genes and selectable marker

29. How Agrobacterium Transforms Plants

30. 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?

31. Select for Transformed Plants
BastaR (green)
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
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)
3. Observe reporter gene expression in T2 seeds at different stages of development under the microscope

32. Where/When Is Your Gene of Interest Transcribed?
TATA box
ATG
STOP
5’ UTR
3’ UTR
Upstream region contains the “ON switch” for your gene of interest
5’ UTR
3’ UTR
GUS
GFP
TATA box RB LB
BastaR
Arabidopsis Silique
Endosperm
Whole Seed
Chalazal Endosperm
GUS
GFP