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Cloning Promoters Kelli Henry April 27, 2009. Mature Green Zygote PreglobularGlobular Transition HeartTorpedoLinear Cotyledon Bending CotyledonLinear.

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Presentation on theme: "Cloning Promoters Kelli Henry April 27, 2009. Mature Green Zygote PreglobularGlobular Transition HeartTorpedoLinear Cotyledon Bending CotyledonLinear."— Presentation transcript:

1 Cloning Promoters Kelli Henry April 27, 2009

2 Mature Green Zygote PreglobularGlobular Transition HeartTorpedoLinear Cotyledon Bending CotyledonLinear Cotyledon Where in the Seed Is Your Gene of Interest Transcribed? At What Stage of Development? Seed Coat Endosperm Embryo

3 What Processes Occur During Seed Development?

4 How Do Genes Get Expressed in a Eukaryote? Seed Leaf 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 SeedChalazal Endosperm Which is more sensitive?

6 Where and When Is Your Gene of Interest Transcribed? TATA box ATGSTOP 5’ UTR 3’ UTR STOP 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 T-DNA Inserts Randomly Into Plant Genome

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

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

13 Where and When Is Your Gene of Interest Transcribed? TATA box ATGSTOP 5’ UTR 3’ UTR STOP 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? TATA box GFP 5’ UTR 3’ UTR GFP TATA box 5’ UTR 3’ UTR Use Directional TOPO Cloning vs

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

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 Extra sequence is cleaved off in E. coli Topoisomerase I recognition sites

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 1.Cleaves and linearizes the pENTR vector, allowing insertion of the PCR fragment 2. Ligates the vector

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

22 How Do You Screen for Recombinant Plasmids with the Insert in the Correct Orientation? Correct orientationIncorrect 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 AT3G05860 promoter in pENTR x SacII x PacI Expected Sizes (bp) Correct Orientation Incorrect Orientation One Cut Vector Only 2600 Isolate plasmid DNA from E. coli colonies and do restriction digest

23 Transfer the Promoter into the T-DNA Shuttle Vector 5’ UTR 3’ UTR GUSGFP TATA box RB LBBasta R

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

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

26 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

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 GUSGFP TATA box RB LBBasta R 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 Basta R (green) Basta S (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)

32 Where/When Is Your Gene of Interest Transcribed? Endosperm Whole SeedChalazal Endosperm GUS GFP Upstream region contains the “ON switch” for your gene of interest TATA box ATGSTOP 5’ UTR3’ UTR STOP 3’ UTR 5’ UTR 3’ UTR GUSGFP TATA box RB LBBasta R Arabidopsis Silique


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