Team:ETH Zurich/Results

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Contents

Results

LovTAP

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.

Lovv.jpg

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 Fig 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.


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.

UVR8

Results

SDS PAGE
Repression studies
Uvrr.jpg

Colonies were cultivated in the dark in the TECAN plate reader during 24 hours and GFP fluorescence intensity was measured during that time. It turned out that the fusion strategy 1 (full UVR8 directly fused to dTetR) represses most in the dark state.

Plans

In a next step we plan to measure also the derepression of the UVR8 dTetR fusion by screening its UV response with the plate reader and carry out single cell analysis by FACS and finally join UVR8 and pABA systems/RecA and show increased E.coli UV-B tolerance (sun cream properties).

UVR8 fusion purification: In vitro studies

Find constants for: Dimerization Monomerization DNA binding etc.


p-ABA generator

Results

agarose gel
Eth ladder paba.jpg
Ladder, A NheI/PstI, A XbaI/PstI, B NheI/PstI, B XbaI/PstI

As the promoter <partinfo>BBa_J23100</partinfo> has two NheI restriction sites, construct A is cut twice (lane 2) while the promoterless construct B is cut only once (lane 4).

Plans

Cloning of B downstream of 1. the Tet promoter and 2. the PL dual input promoter <partinfo>BBa_K909011</partinfo> containing the TetO1 and LacO1 operator sites.





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