Cloning Promoters Kelli Henry April 27, 2009

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

What Processes Occur During Seed Development?

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 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? 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

How Can Your Promoter of Interest Be Isolated and Amplified?

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 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

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

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

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

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

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

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

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’ orientation ATG TAC Promoter of Interest

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.

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 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

How Can Your Promoter of Interest Be Isolated and Amplified?

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

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

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

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

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

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

How Agrobacterium Transforms Plants

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 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

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