Team:Harvard/flavor/results

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<div>Figure 1 &nbsp; <a href="https://static.igem.org/mediawiki/2010/7/77/YFP_Fig_1-crop.jpg" id="single_image" style="font-size:12px">click to enlarge</a></div><hr/>
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<div>Figure 2 &nbsp; <a href="https://static.igem.org/mediawiki/2010/a/af/Western_Fig_2-crop.jpg" id="single_image" style="font-size:12px">click to enlarge</a></div><hr/>
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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 2. Western Blot of Miraculin and Brazzein Expression 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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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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Revision as of 06:01, 25 October 2010



Results

Confirmation with YFP-2x Tags

In order to confirm that the Miraculin and Brazzein are able to be expressed in E. Coli we attached a YFP-2x 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

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. YFP-2x 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 E. Coli

Confirmation with Western Blot

A western blot assay was performed to check for E. Coli expression of

Figure 2   click to enlarge


Figure 2. Western Blot of Miraculin and Brazzein Expression in E. Coli
Proteins were tagged using a StrepII 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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