Part:BBa_K2100049:Experience

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Applications of BBa_K2100049

The MIT iGEM team used pENTR attB-flipped eYFP-attP to create hEF1a: attB-flipped eYFP-attP.

In order to characterize this entry vector, we transfected hEF1a: attB-flipped eYFP-attP into HEK293 cells, along with a constitutively expressed red fluorescence marker. We do not want any leaky expression of this gene because we want to distinguish between the on and off states of TP901 in order to provide a correct diagnosis for endometriosis. We compared the fluorescence of this part to the fluorescence of hEF1a-SV40: eYFP, which showed high expression of eYFP.

hEF1a: attB-flipped eYFP-attP showed no basal expression of the inverted yellow fluorescent protein.

After characterizing the basal expression of this plasmid, we used it to characterize TP901. When TP901 is produced, it recognizes the recombination sites attB and attP in this entry vector, and it inverts the DNA between these two sites. This means that it will irreversibly change this plasmid into one with eYFP in the "on" state. Using hEF1a: attB-flipped eYFP-attP, we characterized TP901 under a hormone-inducible promoter.

We wanted to compare these results to the characterization curve of EGSH to determine the basal expression of TP901. These results determine the need for a higher-level repression system to decrease the efficiency of TP901. TP901 under inducible promoter EGSH is activated by PonA and constitutively expressed transactivator. When TP901 is produced, it inverts the eYFP expression vector into an "on" state. We induced the cells with six different concentrations of PonA: 0 uM, 0.1 uM, 0.5 uM, 1 uM, 2 uM, and 5 uM.

As shown in the graph, we saw approximately a two-fold difference in yellow fluoresence between the uninduced cells and the cells induced with 2 uM or 5 uM (which seemed to be at saturation). We observed a significant amount of basal activity of TP901 even in the absence of PonA, however, so we concluded that an inducible promoter is too leaky to silence the activity of TP901. This means that when TP901 is present in any small amounts, it will flip the eYFP into an "on" state.

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