Team:ETH Zurich/UVR8/Results

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== TetR<sub>DBD</sub>-UVR8 expression studies ==
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[[File:UVR8 SDS native page.jpg|frameless|350px|right|thumb|Figure 1: UVR8 expression: a) SDS-page of TetR<sub>DBD</sub>-dUVR8 expression. An additional 65 kDa protein appears after introducing a TetR<sub>DBD</sub>-dUVR8 gene into the cells. b) Native page of a purified TetR<sub>DBD</sub>-dUVR8-Histag fusion. In native state, TetR<sub>DBD</sub>-dUVR8 forms a dimer which is split by heat denaturation]]
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We tested whether TetR<sub>DBD</sub>-UVR8 fusions can be expressed in as a proper folded proteins in ''E.coli''. SDS-page analysis clearly shows, that introducing TetR<sub>DBD</sub>-dUVR8 results in expression of 65kDa size protein - the same theoretical size as TetR<sub>DBD</sub>-dUVR8 (fig. 1a). Furthermore, we added 7xHistag to C-terminus of the TetR<sub>DBD</sub>-dUVR8 construct and purified it through Ni affinity chromatography column. However, contrarily to literature <span class='eth_reference'>[Heijde2012]</span>, TetR<sub>DBD</sub>-dUVR8 dimer was not seen in SDS-page gels(data not shown). Nevertheless, dimer is observed in the native page analysis, indicating that ''E.coli'' synthesizes a functional TetR<sub>DBD</sub>-dUVR8.
== Repression studies ==
== Repression studies ==
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{| class='tablenoborder'
 
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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>. ]]
 
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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.  
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.  
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As expected, cells with ''reporter only'' showed high GFP fluorescence, whereas 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. Another explanation would be that TetR can bind P<sub>tet</sub> more efficiently due to cooperativity. If the latter is true, fusion of the TetR<sub>DBD</sub> with proteins, that are able to dimerize, would restore its activity to repress the P<sub>tet</sub> promoter.  
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As expected, cells with ''reporter only'' showed high GFP fluorescence, whereas 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 2). These results can be explained in two ways: dimerizing domain is required for proper TetR<sub>DBD</sub> folding. Another explanation would be that TetR can bind P<sub>tet</sub> more efficiently due to cooperativity. If the latter is true, fusion of the TetR<sub>DBD</sub> with proteins, that are able to dimerize, would restore its activity to repress the P<sub>tet</sub> promoter.  
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[[File:TetRDBD_1.png|frameless|600px|center|thumb|Figure 2: Fluorescence normalized to the OD for wt, reporter only, TetR and TetR<sub>DBD</sub>. TetR shows the fluorescence level as the wildtype.]]
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Thus we fused TetR<sub>DBD</sub> with three versions of UVR8 proteins each varying in linker length. 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 we are able to repress GFP production by fusing TetR<sub>DBD</sub> with UVR8. We limit our further studies to the best performing chimera – TetR<sub>DBD</sub>-dUVR8.
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Thus we fused TetR<sub>DBD</sub> with three versions of UVR8 proteins each varying in linker length ([[Team:ETH_Zurich/UVR8/Design|see Design]]). Again, fluorescence from cells cotransfected with fusion proteins and reporter ([[Team:ETH_Zurich/UVR8/Design#Plasmids|see Plasmids]]) were compared with reporter only and wildtype. Figure 3 shows that we are able to repress GFP production by fusing TetR<sub>DBD</sub> with UVR8. We limit our further studies to the best performing chimera – TetR<sub>DBD</sub>-truncated-UVR8 ([[Team:ETH_Zurich/UVR8/Design|see 3.]]).
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[[File:Repression_1.png|frameless|600px|center|thumb|Figure 2: Comparison of different TetR<sub>DBD</sub>-dUVR8 fusions with TetR and reporter only ]]
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[[File:UVR8_3.png|frameless|600px|center|thumb|Figure 3: Comparison of different TetR<sub>DBD</sub>-UVR8 fusions with TetR and reporter only ]]
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[[File:IPTG_1.png|frameless|600px|center|thumb|Figure 3: Cell growth dependend on UVR8 expression ]]
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[[File:IPTG_1.png|frameless|600px|center|thumb|Figure 4: Cell growth dependent on UVR8 expression ]]
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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.  
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However, high inductions of UVR8 are toxic for cells and impair cell growth (fig. 4). Nevertheless, for our purposes we do not need high overexpression of the protein thus we tested repression dependency on protein induction.  
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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).   
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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. 5 and fig. 6).   
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[[File:IPTG.png|frameless|500px|center|thumb|Figure 4: Repression depended on UVR8 concentration]]
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[[File:UVR8_4.png|frameless|600px|center|thumb|Figure 5: Repression dependent on TetR-DBD-UVR8 induction via different IPTG concentrations. left: reporter only as negative control]]
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[[File:IPTG_2.png|frameless|500px|center|thumb|Figure 5: ]]
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[[File:UVR8_5.png|frameless|600px|center|thumb|Figure 6: Cells expressing TetR<sub>DBD</sub>-UVR8 grow at low inducer IPTG concentrations, thus low TetR-DBD-UVR8 concentration]]
== New part TetR<sub>DBD</sub> ==
== New part TetR<sub>DBD</sub> ==
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We showed that the TetR monomer is unable to repress the P<sub>tet</sub> 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 P<sub>tet</sub> promoter and any reporter gene, one can test whether certain proteins are able to dimerize in a cell or not.
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We showed that the TetR monomer is unable to repress the P<sub>tet</sub> 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 P<sub>tet</sub> promoter and any reporter gene, one can test whether certain proteins are able to [[Team:ETH_Zurich/UVR8#TetRDBD_-_a_novel_two-hybrid_screening_in_E.coli|dimerize]] in a cell or not.
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==Achievements so far==
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* TetR<sub>DBD</sub> only binds to DNA as a dimer
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** enables to use TetR<sub>DBD</sub> in a two-hybrid screening or as a switch
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* TetR<sub>DBD</sub>-UVR8 fusion is able to act as a repressor
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== Plans ==
 
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In a next step we plan to measure also the derepression of the UVR8-TetR<sub>DBD</sub> 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).
 
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*UVR8 fusion purification: ''In vitro'' studies
 
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*Find constants for dimerization, monomerization DNA binding etc.
 
{{:Team:ETH_Zurich/Templates/Footer}}
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Latest revision as of 02:46, 27 October 2012

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Navigate through Overview, Design and Results

Contents

TetRDBD-UVR8 expression studies

Figure 1: UVR8 expression: a) SDS-page of TetRDBD-dUVR8 expression. An additional 65 kDa protein appears after introducing a TetRDBD-dUVR8 gene into the cells. b) Native page of a purified TetRDBD-dUVR8-Histag fusion. In native state, TetRDBD-dUVR8 forms a dimer which is split by heat denaturation

We tested whether TetRDBD-UVR8 fusions can be expressed in as a proper folded proteins in E.coli. SDS-page analysis clearly shows, that introducing TetRDBD-dUVR8 results in expression of 65kDa size protein - the same theoretical size as TetRDBD-dUVR8 (fig. 1a). Furthermore, we added 7xHistag to C-terminus of the TetRDBD-dUVR8 construct and purified it through Ni affinity chromatography column. However, contrarily to literature [Heijde2012], TetRDBD-dUVR8 dimer was not seen in SDS-page gels(data not shown). Nevertheless, dimer is observed in the native page analysis, indicating that E.coli synthesizes a functional TetRDBD-dUVR8.


Repression studies

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 reporter only showed high GFP fluorescence, whereas 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 2). These results can be explained in two ways: dimerizing domain is required for proper TetRDBD folding. Another explanation would be that TetR can bind Ptet more efficiently due to cooperativity. If the latter is true, fusion of the TetRDBD with proteins, that are able to dimerize, would restore its activity to repress the Ptet promoter.

Figure 2: Fluorescence normalized to the OD for wt, reporter only, TetR and TetRDBD. TetR shows the fluorescence level as the wildtype.


Thus we fused TetRDBD with three versions of UVR8 proteins each varying in linker length (see Design). Again, fluorescence from cells cotransfected with fusion proteins and reporter (see Plasmids) were compared with reporter only and wildtype. Figure 3 shows that we are able to repress GFP production by fusing TetRDBD with UVR8. We limit our further studies to the best performing chimera – TetRDBD-truncated-UVR8 (see 3.).


Figure 3: Comparison of different TetRDBD-UVR8 fusions with TetR and reporter only


Figure 4: Cell growth dependent on UVR8 expression


However, high inductions of UVR8 are toxic for cells and impair cell growth (fig. 4). 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. 5 and fig. 6).


Figure 5: Repression dependent on TetR-DBD-UVR8 induction via different IPTG concentrations. left: reporter only as negative control
Figure 6: Cells expressing TetRDBD-UVR8 grow at low inducer IPTG concentrations, thus low TetR-DBD-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 whether certain proteins are able to dimerize in a cell or not.

Achievements so far

  • TetRDBD only binds to DNA as a dimer
    • enables to use TetRDBD in a two-hybrid screening or as a switch
  • TetRDBD-UVR8 fusion is able to act as a repressor




References

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