Cloning Promoters Figure 20-25 Figure 20-25

Cloning Promoters Figure 20-25 Figure 20-25 www.phwiki.com

Cloning Promoters Figure 20-25 Figure 20-25

Brickman, Jim, Host has reference to this Academic Journal, PHwiki organized this Journal Cloning Promoters Kelli Henry April 27, 2009 Where in the Seed Is Your Gene of Interest Transcribed At What Stage of Development Seed Coat Endosperm Embryo What Processes Occur During Seed Development

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How Do Genes Get Expressed in a Eukaryote Seed Leaf Do Arabidopsis leaves in addition to 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 in addition to When Is Your Gene of Interest Transcribed TATA box ATG STOP 5’ UTR 3’ UTR STOP 3’ UTR

How Can Your Promoter of Interest Be Isolated in addition to Amplified How Can Plant Cells Be Trans as long as med What is the structure of a plant cell Do plant cells have plasmids How can you take advantage of a natural process to trans as long as m a plant How to Trans as long as m 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 Trans as long as ms Plants T-DNA Inserts R in addition to omly Into Plant Genome Figure 20-25 Agrobacterium Ti-Plasmid ~5kb For Your Vector What Is Totipotency How does A. tumefaciens infect a plant

Where in addition to When Is Your Gene of Interest Transcribed TATA box ATG STOP 5’ UTR 3’ UTR STOP 3’ UTR How Can You Get the Promoter to Insert into a Vector in the Correct Orientation TATA box GFP 5’ UTR 3’ UTR GFP Use Directional TOPO Cloning vs 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

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

What Is Topoisomerase I Topoisomerase I relieves supercoils in circular DNA plasmids by nicking one of the str in addition to s of the DNA double helix, twisting it around the other str in addition to , in addition to re-ligating the nicked str in addition to 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 in addition to linearizes the pENTR vector, allowing insertion of the PCR fragment 2. Ligates the vector How Can Your Promoter of Interest Be Isolated in addition to Amplified

How Do You Screen as long as 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 Transfer the Promoter into the T-DNA Shuttle Vector Transfer the Promoter into the T-DNA Shuttle Vector Use Shuttle vector in addition to Helper Ti plasmid instead of Ti plasmid

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Figure 20-25 Agrobacterium Ti-Plasmid ~5kb For Your Vector How Can the Promoter Be Transferred from pENTR Vector to the T-DNA Vector + + LR Clonase™ II 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 in addition to opines (a carbon/nitrogen source that can only be metabolized by Agrobacterium) between LB in addition to RB How Agrobacterium Trans as long as ms Plants Dip Arabidopsis in a Solution of Agrobacterium Containing Your T-DNA Floral dipping trans as long as ms the female reproductive tissues that give rise to seeds after fertilization. ~1% of seeds are trans as long as med. How do you select as long as trans as long as med seeds

Select as long as Trans as long as med 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

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