Template:Nevada/TheCoronatineSystem

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                                    <img src="https://static.igem.org/mediawiki/2014/c/c1/Team_nevada_research_verifying_coi1_and_ha_in_plasmid.png" alt="Post Image" />
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                                            <a  title="Amplification of JAZ degron tag and plasmid provided by UCSF iGEM ">
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                                                <b>Amplification of JAZ degron tag and plasmid provided by UCSF iGEM </b>
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                                        <p>JAZ1 has already been proven to be a successful degron tag in yeast. We are using this as a positive control while also researching the efficiency of JAZ6 as a degron in yeast. The degron tags were PCR amplified and verified through gel electrophoresis. The UCSF iGEM team also provided a pSV606 plasmid containing the pTEF constitutive promoter and GFP. Through designed primers, we again amplified only the pTEF and GFP out of the plasmid in the interest of time.    </p>
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Revision as of 23:09, 17 October 2014

APP ON Blog

Team Nevada

The BAITswitch

The Bioorthoginal Auxin Induceable Trigger Switch
Post Image

Amplification of E3 ubiquitin ligase

COI1 is the E3 ubiquitin ligase for the JAZ degron pathway. This is analogous to the TIR1 enzyme in the auxin pathway, but instead it binds coronatine. Normally, COI1 binds JA-Ile (jasmonic acid-isoleucine) but this compound is difficult and expensive to synthesize and cannot be purchased. Coronatine is structurally similar and is manufactured, which is why we used it instead. COI1 was amplified and verified through gel electrophoresis.

Post Image

Gibson Assembly and E. coli transformation

Just like we tagged TIR1 with Myc, here we tagged COI1 with an HA tag for screening. Amplified and tagged COI1 was then joined to pTEF-GFP, inserted into a bacterial vector and transformed with NEB 10 beta competent cells with LB-ampicillin selection. You may have noticed that we use GFP in both of our systems, though we said our goal was a bioorthogonal system to degrade two proteins simultaneously. At the beginning, our use of RFP was not very successful, so for the interest of making sure we can build a construct that works, we used what we had which was GFP and will later swap it out for mCherry when we juxtapose the systems into yeast.

Integration of Engineered Vectors into Yeast Cells

After the above construct grew successful colonies, we isolated and purified the newly engineered vector and transformed into W303 yeast cells. It was screened via yeast colony PCR.

Confirmation of COI1 expression in yeast

Protein was extracted from yeast via alkali treatment and analyzed on a Western blot and Coomassie blue assay. .

Post Image

Amplification of JAZ degron tag and plasmid provided by UCSF iGEM

JAZ1 has already been proven to be a successful degron tag in yeast. We are using this as a positive control while also researching the efficiency of JAZ6 as a degron in yeast. The degron tags were PCR amplified and verified through gel electrophoresis. The UCSF iGEM team also provided a pSV606 plasmid containing the pTEF constitutive promoter and GFP. Through designed primers, we again amplified only the pTEF and GFP out of the plasmid in the interest of time.

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