Cloning Promoters

1. Cloning Promoters Kelli Henry April 27, 2009

2. 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

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. 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

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

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

13. 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

14. 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

15. 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

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 CloningA 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

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 2. Ligates the vector

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

22. 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

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

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

25. Figure 20-25 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. 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

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. 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

32. 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?