Template:Nevada/TheCoronatineSystem

From 2014.igem.org

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<p> <b> Synthesis and Engineering of the Coronatine Pathway Components </b></p>
 
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                                     <img src="https://static.igem.org/mediawiki/2014/d/d6/Team_nevada_research_amplifying_coi1.png" alt="Post Image" />
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                                     <img src="https://static.igem.org/mediawiki/2014/d/d0/Team_nevada_research_jaz_1_and_jaz_6_verification.png" alt="Post Image" />
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                                            <a  title="Integration of Engineered Vectors into Yeast Cells">
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                                                <b>Integration of Engineered Vectors into Yeast Cells</b>
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                                            </a>
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                                        <p>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.</p>
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                                             <a  title="Confirmation of COI1 expression in yeast">
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                                                 <b>Integration of Engineered Vectors into Yeast Cells</b>
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                                                 <b>Confirmation of COI1 expression in yeast</b>
                                             </a>
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                                         <p>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.</p>
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                                         <p>Protein was extracted from yeast via alkali treatment and analyzed on a Western blot and Coomassie blue assay. .</p>
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                                             <a  title="Amplification of JAZ degron tag and plasmid provided by UCSF iGEM ">
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                                                 <b>Confirmation of COI1 expression in yeast</b>
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                                                 <b>Amplification of JAZ degron tag and plasmid provided by UCSF iGEM </b>
                                             </a>
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                                         <p>Protein was extracted from yeast via alkali treatment and analyzed on a Western blot and Coomassie blue assay. </p>
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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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                                            <a  title="Gibson Assembly and 2μ transformation">
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                                                <b>Gibson Assembly and 2μ transformation</b>
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                                        <p>pTEF-GFP and the JAZ degrons were cloned and transformed into a 2μ plasmid. Like our auxin pathway, this would then be introduced to the COI1 plasmid and we would continue with our experiments of adding varying concentrations of coronatine.  </p>
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                                    <img src=" https://static.igem.org/mediawiki/2014/f/f4/Team_nevada_research_verifying_gfp_in_yeast.png" alt="Post Image" />
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                                            <a  title="Confirmation of coronatine-induced degron system via Western Blot densitometry and fluorescence analysis ">
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                                                <b>Confirmation of coronatine-induced degron system via Western Blot densitometry and fluorescence analysis</b>
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                                            </a>
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                                        </h2>
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                                        <p>After the introduction of coronatine, the degradation of GFP was monitored using a spectrophotometer as well as Western Blot densitometry. Because coronatine is to induce the destruction of GFP, we would expect to not see GFP on the Western blot and to only see a band on our positive, bacterial control.  </p>
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Latest revision as of 23:20, 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.

Gibson Assembly and 2μ transformation

pTEF-GFP and the JAZ degrons were cloned and transformed into a 2μ plasmid. Like our auxin pathway, this would then be introduced to the COI1 plasmid and we would continue with our experiments of adding varying concentrations of coronatine.

Post Image

Confirmation of coronatine-induced degron system via Western Blot densitometry and fluorescence analysis

After the introduction of coronatine, the degradation of GFP was monitored using a spectrophotometer as well as Western Blot densitometry. Because coronatine is to induce the destruction of GFP, we would expect to not see GFP on the Western blot and to only see a band on our positive, bacterial control.

Retrieved from "http://2014.igem.org/Template:Nevada/TheCoronatineSystem"