Team:ETH Zurich/LovTAP/Results

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Results

We tested the LovTap construct with RFP as an output. Light activated LovTap is supposed to act as a repressor at the Trp operator and therefore we would expect a reduction of the RFP signal. Measurements with the plate reader lead to confusing results, therefore we switch to single cell analysis with FACS. The results show a decrease of RFP the first 5 hours, which might be due to the inoculation and RFP maturation.

Figure 1: Here we show the fluorescence intensity over time. As an output of the FACS analysis we used the median of fluorescence. We compared two conditions: light and no light. As a control we used the wild-type and reporter only.


If we compare the signal strength of the negative control: reporter only, in the two different conditions, light and no light, we are not expecting a decrease of RFP. The decrease of RFP shown in the figure is a result of bleaching the RFP. Also for the LovTap construct we see a different output between the two conditions, but this is most probably also due to bleaching of the RFP with the light we use to activate LovTap.


Plans

Since we are not able to get reliable results due to bleaching, we are switching our read-out system so that we will have a Galactosidase as an output. As a next step we will measure the LovTap construct in the same conditions using a Miller assay. Also an important change we would like to make is to invert the signal output, which means that we are not repressing our output with blue light but activating it instead (see constructs). We are working on cloning this construct and will test it soon.


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