Team:Harvard/flavor/results

From 2010.igem.org

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The two flavors that are currently ready for transformation into plants are the "taste-inverter" <u>miraculin</u> and the sweetener <u>brazzein</u>.  Given the long time-frame of plant transformation we used two different assays in <i>E. Coli</i> to confirm that our proteins could indeed be transcribed and translated. The results of those assays are shown here.
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The two flavors that are currently ready for transformation into plants are the "taste-inverter" <a href="http://en.wikipedia.org/wiki/Miraculin">miraculin</a> and the sweetener <a href="http://en.wikipedia.org/wiki/Brazzein">brazzein</a>.  Given the long time-frame of plant transformation we used two different assays in <i>E. Coli</i> to confirm that our proteins could indeed be transcribed and translated. The results of those assays are shown here.
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<h2>Confirmation with <i>YFP-2x</i> Tags</h2>
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<h2>confirmation with 2xYFP tags</h2>
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In order to confirm that the Miraculin and Brazzein are able to be expressed in <i>E. Coli</i> we attached a <i>YFP-2x</i> tag sequence to the termini of both proteins.  The proteins were placed under an IPTG-expressible promoter and used spectrophotometry to determine the level of YFP fluorescence against a baseline, untagged protein.  Figure 1 shows relative-fluorescence at times post induction.  In all circumstances the levels of YFP-fluorescence increased.
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In order to confirm that miraculin and brazzein are able to be expressed in <i>E. Coli</i>, we attached a 2xYFP tag sequence to both the N- and C-termini of each protein.  The proteins were placed under an IPTG-expressible promoter, and spectrophotometry was used to determine the level of YFP fluorescence against a baseline, untagged protein.  Figure 1 shows relative-fluorescence at times post induction.  In all circumstances the levels of YFP-fluorescence increased.
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<u>Figure 1. Induced expression of YFP-tagged Miraculin and Brazzein in E. Coli</u></br>
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<u>Figure 1. Induced expression of YFP-tagged Miraculin and Brazzein in <i>E. Coli</i></u></br>
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Figure 1 (A) through (D) are normalized plots of Miraculin and Brazzein YFP-fused constructs expressed in E. Coli. <i>YFP-2x</i> tags were attached to both N- and C- termini to ensure that folding was not hindered.  In all cases relative YFP fluorescence had appreciably increased after 120 minutes as compared to the non-induced <i>E. Coli</i>
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Figure 1 (A) through (D) are normalized plots of miraculin and brazzein YFP-fused constructs expressed in E. Coli. 2xYFP tags were attached to either N- or C- terminus to ensure that folding was not hindered.  In all cases relative YFP fluorescence had appreciably increased after 120 minutes as compared to the non-induced <i>E. Coli</i>
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<h2>Confirmation with Western Blot</h2>
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<h2>confirmation by western blot</h2>
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<p>
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A western blot assay was performed to check for <i>E. Coli</i> expression of Miraculin and Brazzein.  Proteins tagged at either the N- or C- terminus were placed under the control of an IPTG-inducible promoter.  In the miraculin assay, no protein expression was seen.  It is possible that the protein does not express well in <i>E. Coli</i>, or that the plant-specific codon optimization of the proteins resulted in reduced expressibility.  Brazzein, specifically C-terminus tagged brazzein was seen to be highly expressed in <i>E. Coli</i>.
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A western blot assay was performed to check for <i>E. Coli</i> expression of miraculin and brazzein.  Proteins tagged at either the N- or C- terminus were placed under the control of an IPTG-inducible promoter.  In the miraculin assay, no protein expression was seen.  It is possible that the protein does not express well in <i>E. Coli</i>, or that the plant-specific codon optimization of the proteins resulted in reduced expressibility.  Brazzein, specifically C-terminus tagged brazzein was seen to be highly expressed in <i>E. Coli</i>.
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<u>Figure 2. Western Blot of Miraculin and Brazzein Expression in <i>E. Coli</i></u></br>
<u>Figure 2. Western Blot of Miraculin and Brazzein Expression in <i>E. Coli</i></u></br>
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Proteins were tagged using a <i>StrepII</i> standard Antibody tag, attached to both N- and C- termini. Miraculin (A) does not appear to have been expressed in high enough quantities to be visualized. The expected protein weight is 25 kDa.  Brazzein (B) shows strong expression of a protein in the 10-15 kDa range.  Brazzein has an expected weight of 6.5 kDa, a discrepancy that we have attributed to inconsistencies in the gel.
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Proteins were tagged using a standard <i>StrepII</i> tag, attached to either the N- or C- terminus. Miraculin (A) does not appear to have been expressed in high enough quantities to be visualized. The expected protein weight is 25 kDa.  Brazzein (B) shows strong expression of a protein in the 10-15 kDa range.  Brazzein has an expected weight of 6.5 kDa, a discrepancy that we have attributed to inconsistencies in the gel.
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<h2>Expression in Arabidopsis</h2>
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<h2>expression in <i>Arabidopsis</i></h2>
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<p>We are still waiting for the plants to grow to a size large enough that we can collect samples to verify expression, but we have selected for plants that have integrated the herbicide resistance marker along with the miraculin and brazzein expression constructs.</p>
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<p>We are still waiting for the plants to grow to a size large enough that we can collect samples to verify expression, but we have selected for plants that have integrated the glufosinate resistance marker along with the miraculin and brazzein expression constructs.</p>
<div><strong>Miraculin:</strong> <a href="https://static.igem.org/mediawiki/2010/5/53/V21selection.jpg" id="single_image" style="font-size:12px">click to enlarge</a></div>
<div><strong>Miraculin:</strong> <a href="https://static.igem.org/mediawiki/2010/5/53/V21selection.jpg" id="single_image" style="font-size:12px">click to enlarge</a></div>

Latest revision as of 00:19, 27 October 2010



confirmation results

The two flavors that are currently ready for transformation into plants are the "taste-inverter" miraculin and the sweetener brazzein. Given the long time-frame of plant transformation we used two different assays in E. Coli to confirm that our proteins could indeed be transcribed and translated. The results of those assays are shown here.

confirmation with 2xYFP tags

In order to confirm that miraculin and brazzein are able to be expressed in E. Coli, we attached a 2xYFP tag sequence to both the N- and C-termini of each protein. The proteins were placed under an IPTG-expressible promoter, and spectrophotometry was used to determine the level of YFP fluorescence against a baseline, untagged protein. Figure 1 shows relative-fluorescence at times post induction. In all circumstances the levels of YFP-fluorescence increased.

Figure 1   click to enlarge


Figure 1. Induced expression of YFP-tagged Miraculin and Brazzein in E. Coli
Figure 1 (A) through (D) are normalized plots of miraculin and brazzein YFP-fused constructs expressed in E. Coli. 2xYFP tags were attached to either N- or C- terminus to ensure that folding was not hindered. In all cases relative YFP fluorescence had appreciably increased after 120 minutes as compared to the non-induced E. Coli

confirmation by western blot

A western blot assay was performed to check for E. Coli expression of miraculin and brazzein. Proteins tagged at either the N- or C- terminus were placed under the control of an IPTG-inducible promoter. In the miraculin assay, no protein expression was seen. It is possible that the protein does not express well in E. Coli, or that the plant-specific codon optimization of the proteins resulted in reduced expressibility. Brazzein, specifically C-terminus tagged brazzein was seen to be highly expressed in E. Coli.

Figure 2   click to enlarge


Figure 2. Western Blot of Miraculin and Brazzein Expression in E. Coli
Proteins were tagged using a standard StrepII tag, attached to either the N- or C- terminus. Miraculin (A) does not appear to have been expressed in high enough quantities to be visualized. The expected protein weight is 25 kDa. Brazzein (B) shows strong expression of a protein in the 10-15 kDa range. Brazzein has an expected weight of 6.5 kDa, a discrepancy that we have attributed to inconsistencies in the gel.

expression in Arabidopsis

We are still waiting for the plants to grow to a size large enough that we can collect samples to verify expression, but we have selected for plants that have integrated the glufosinate resistance marker along with the miraculin and brazzein expression constructs.

Miraculin: click to enlarge



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