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Testing TP901 under the inducible promoter EGSH

System Mechanism

EGSH is an inducible promoter that behaves similarly to the TRE promoter: when its transactivator, the ecdysone receptor (VgEcR) binds to a small molecule called ponasterone A (PonA), it will bind to the EGSH promoter and initiate transcription of the gene downstream from the promoter. In this experiment, that gene is TP901, a serine integrase. Our motivation for using the EGSH/PonA system instead of the TRE/doxycyline system is that as its name suggests, ponasterone A is a hormone, so we expect this system to behave more similarly to our synthetic estrogen and progesterone responsive promoter systems than TRE/doxycycline. To measure the activity of TP901, we used Golden Gate assembly to flank an inverted gene for enhanced yellow fluorescent protein (eYFP) with the attB and attP recognition sites for TP901 and, using gateway cloning, put it under the constitutive mammalian promoter human elongation factor 1-alpha (hEF1a). Thus, the unmodified expression vector does not produce any eYFP, but if TP901 is present and active, it will unidirectionally invert the gene to the correct orientation for the promoter, and the cells will express eYFP.

Experiment 1: Characterizing EGSH Promoter

1. Purpose

2. Set Up

3. Results

Experiment 2: Testing the Flipped Gene vs. Transcriptional Stop Signal

1. Purpose

2. Set Up

3. Results

Experiment 3: Inducible TP901

1. Purpose

2. Set Up

We cotransfected the EGSH: TP901 and hEF1a: attB-flipped EYFP-attP expression vectors along with EGSH: mKate (to indicate how much TP901 the cells were expressing), hEF1a: VgEcR (the transactivator for EGSH), and hEF1a: BFP (a transfection marker) into HEK293 cells. 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.

3. Results

NOT SURE WHY AXIS TITLES GOT SWTICHED BUT NEED TO FIX THIS As shown in the bar 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 issue was our motivation for exploring the L7Ae/k-turn motif as a way to lower the basal expression of TP901, and we hope that this system will allow us to inhibit recombination when the system is uninduced but not when it is activated.