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Team:Harvard/flavor/results
From 2010.igem.org
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.
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Figure 1 click to enlarge 
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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.
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Figure 2 click to enlarge 
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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.
