Team:ETH Zurich/UVR8/Results

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[[File:TetRDBD_1.png|frameless|450px|right|thumb|Figure 1: Fluorescence normalized to the OD for wt, reporter only, TetR and TetR<sub>DBD</sub>. ]]
[[File:TetRDBD_1.png|frameless|450px|right|thumb|Figure 1: Fluorescence normalized to the OD for wt, reporter only, TetR and TetR<sub>DBD</sub>. ]]
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In order to test whether a UVR8 fusion with the TetR DNA binding domain (TetR<sub>DBD</sub>) is reasonable, we first had to test, if our used TetR<sub>DBD</sub> is not able to repress the expression of GFP that is controlled by a P<sub>tet</sub> promoter. To test this we measured fluorescence from three different cultures: reporter only, reporter co-transfected with full length TetR or TetR<sub>DBD</sub>. Cells without any plasmid were used as a background fluorescence control.  
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In order to test whether a UVR8 fusion with the TetR DNA binding domain (TetR<sub>DBD</sub>) is reasonable, we first had to test, if our used TetR<sub>DBD</sub> is not able to repress the expression of GFP that is controlled by a P<sub>tet</sub> promoter. For this purpose we measured the fluorescence from three different cultures: reporter only, reporter co-transfected with full length TetR or TetR<sub>DBD</sub>. Cells without any plasmid termed ''WT'' were used as a background fluorescence control.  
As expected, cells with just reporter plasmid showed high GFP fluorescence output, where as cells containing full length tetR have a tightly repressed GFP production. In addition, cells expressing TetR<sub>DBD</sub> also express GFP at similar levels to those with just the reporter plasmid (see figure 1). These results can be explained in two ways: dimerizing domain is required for proper TetR<sub>DBD</sub> folding. An other explanation would be that TetR can bind P<sub>tet</sub> more efficiently due to cooperativity. If the later is true, fusion of the TetR<sub>DBD</sub> with proteins, which form dimers, would restore its activity to repress the P<sub>tet</sub> promoter.  
As expected, cells with just reporter plasmid showed high GFP fluorescence output, where as cells containing full length tetR have a tightly repressed GFP production. In addition, cells expressing TetR<sub>DBD</sub> also express GFP at similar levels to those with just the reporter plasmid (see figure 1). These results can be explained in two ways: dimerizing domain is required for proper TetR<sub>DBD</sub> folding. An other explanation would be that TetR can bind P<sub>tet</sub> more efficiently due to cooperativity. If the later is true, fusion of the TetR<sub>DBD</sub> with proteins, which form dimers, would restore its activity to repress the P<sub>tet</sub> promoter.  

Revision as of 19:58, 26 September 2012

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Navigate through Overview, Design and Results


Contents

Repression studies

Figure 1: Fluorescence normalized to the OD for wt, reporter only, TetR and TetRDBD.

In order to test whether a UVR8 fusion with the TetR DNA binding domain (TetRDBD) is reasonable, we first had to test, if our used TetRDBD is not able to repress the expression of GFP that is controlled by a Ptet promoter. For this purpose we measured the fluorescence from three different cultures: reporter only, reporter co-transfected with full length TetR or TetRDBD. Cells without any plasmid termed WT were used as a background fluorescence control.

As expected, cells with just reporter plasmid showed high GFP fluorescence output, where as cells containing full length tetR have a tightly repressed GFP production. In addition, cells expressing TetRDBD also express GFP at similar levels to those with just the reporter plasmid (see figure 1). These results can be explained in two ways: dimerizing domain is required for proper TetRDBD folding. An other explanation would be that TetR can bind Ptet more efficiently due to cooperativity. If the later is true, fusion of the TetRDBD with proteins, which form dimers, would restore its activity to repress the Ptet promoter.



Thus we fused TetRDBD with three versions of UVR8 proteins, which varied in linker length between TetRDBD and UVR8. Again, fluorescence from cells having fusion proteins containing plasmid together with a reporter plasmid was compared with reporter only and wildtype. Fig.2 shows that by fusing TetRDBD with UVR8 dimer, we can repress GFP production from Ptet promoter. Furthermore, we limited our studies to the best performing chimera – TetRDBD-dUVR8.


Figure 2: Comparison of different TetRDBD-dUVR8 fusions with TetR and reporter only


Figure 3: Cell growth dependend on UVR8 expression


However, high inductions of UVR8 are toxic for cells and impair cell growth (fig.3). Nevertheless, for our purposes we do not need high overexpression of the protein thus we tested repression dependency on protein induction. Since the expression of UVR8 is induced by IPTG, we varied IPTG concentrations. The results show that even small amounts, 0.01-0.025 mM, of inducer results in GFP repression which does not impair cell growth (fig.4).


Figure 4: Repression depended on UVR8 concentration


New part TetRDBD

We showed that the TetR monomer is unable to repress the PTet promoter. This phenomenon makes our new part [http://partsregistry.org/Part:BBa_K909007 BBa_K909007] a powerful tool. Any protein can be fused in case the coding DNA contains a BamHI restriction site. By that fusion, any protein able to dimerize acts as a transcriptional repressor. By combining the PTet promoter and any reporter gene, one can test if certain proteins are able to dimerize in a cell or not.



Plans

In a next step we plan to measure also the derepression of the UVR8-TetRDBD fusion by screening its UV response with the plate reader and carry out single cell analysis by FACS and finally join UVR8 and p-ABA 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.


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