Experiments Characterizing Recombinase Models
We set out to characterize a recombinase, the serine integrase TP901, under an inducible promoter in order to create a system memory for our temporally specific diagnostic tool.
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 this gene entry vector under the constitutive mammalian promoter human elongation factor 1-alpha (hEF1a). The unmodified expression vector does not produce any eYFP because the gene is upside-down and backwards, but if TP901 is present and active, it will unidirectionally invert the flipped eYFP gene to the correct orientation for the promoter, and the cells will express yellow fluorescence.
We also assembled an output plasmid containing a transcriptional stop signal, SV40, preceding the yellow fluorescent protein output. We could use an excision recombinase, such as Cre or FLP, to cut out this stop signal, and thus express yellow fluorescence. When compared to the first plasmid, however, this model showed high basal fluorescence, and thus we continued our experiments with TP901 instead of Cre or FLP.
Our results showed that the fluorescent protein inverted to an "off" state showed no basal expression, so we used the flipping function of TP901 to turn on this protein and give a readout. After successfully cloning our plasmids for the recombinase recognition sites, we transiently transfected them into HEK293 cells and analyzed the data with flow cytometry.
Experiment 1: Characterizing EGSH Ponasterone A inducible promoter
Mechanism of EGSH Ponasterone A (PonA) system
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 of 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 like our synthetic estrogen and progesterone responsive promoter systems than TRE/doxycycline.
The purpose of this experiment was to characterize the PonA-inducible promoter, pEGSH, which we later used to test how an inducible promoter can modulate the activity of recombinases, ideally keeping basal expression to a minimum. The results of this experiment show us a response curve of the promoter. We also wanted to determine the concentration of PonA that gave the highest amount of EGSH activation compared to the basal levels.
2. Set Up
(1) Constitutively expressed transactivator VgEcR-2A-RXR binds to (2) pEGSH in the presence of small molecule PonA, activating expression of mKate fluorescent protein. (3) Constitutively expressed BFP acts as the transfection marker.
We used a 3:1:1 ratio of EGSH:mKate, transactivator hEF1a:VgEcR, and transfection marker hEF1a:BFP. The 2014 MIT iGEM team, who also used EGSH as an inducible promoter, reported seeing the greatest success with this ratio. We induced cells containing these plasmids with 5 different amounts of PonA. We also added a control sample of HEK293 lacking the transactivator necessary for EGSH. The purpose of this control was to further characterize any leaky expression of the EGSH promoter. The transfection marker, hEF1a:BFP, is a constitutively expressed fluorescent protein, which indicates how many copies of the plasmid a particular cell has. It allows us to analyze data by comparing transfection levels to amount of output.
Results for our induction of EGSH promoter. Color facet represents different amounts of PonA. We saw the greatest level of induction with 5.0 uM PonA.
We observed a two-fold increase between induced and uninduced EGSH, but we noticed a decent amount of basal expression with no PonA added. The saturation occurred around 5 uM of PonA, so we determined that this concentration is the best amount of PonA to compare on/off states of the promoter in our next experiments.
Experiment 2: Testing the Flipped Gene vs. Transcriptional Stop Signal
The purpose of this experiment was to make sure that the plasmids that we designed and built do not express the gene of interest, in our case a yellow fluorescent protein, when there is no recombinase present. 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.
The results of this experiment determined how we would continue. If the transcriptional stop signal repressed yellow fluorescence, we would characterize excision recombinases Cre and FLP. If the flipped yellow fluorescent protein showed no basal expression, we would focus on experiments with serine integrate TP901.
2. Set Up
Two experimental models we transfected into separate wells to determine basal expression. Constitutively active BFP acts as our transfection marker.
We transfected one sample with a plasmid containing a transcriptional stop signal in front of a fluorescent protein, expressed under a strong constitutive promoter, along with a transfection marker plasmid. Another sample contained a plasmid with an upside down fluorescent protein flanked by recombinase recognition sites, expressed under a strong constitutive promoter, along with a transfection marker plasmid.
The green line represents model (a) with the inverted eYFP gene, while the red line represents model (b) containing the stop signal.
We observed no basal yellow fluorescence of the flipped eYFP gene, and high basal expression of the eYFP gene preceded by the transcriptional stop signal. Thus, we decided to continue our TP901 characterization using the flipped eYFP gene.
Experiment 3: Inducible TP901
The purpose of this experiment was to characterize activation of 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.
2. Set Up
We cotransfected the EGSH:TP901 and hEF1a:attB-flipped eYFP-attP (from experiment 2) expression vectors, along with constitutively expressed transactivator and transfection marker hEF1a:BFP.
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.
Results for our inducible TP901 experiment. Color facet represents different amounts of PonA. We saw a two-fold difference in induction between on and off states.
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 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.
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- Breuner, Brondstead, Hammer. Resolvase-like recombination performed by the TP901-1 integrase, Microbiology Society 147: 2051-2063 (2001)