Team:Trieste/parts/3

From 2012.igem.org

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<h2>Description </h2>  
<h2>Description </h2>  
This composite produces constitutively the CymR regulator protein, that binds the Cumate Operator repressing the transcription from the promoter.
This composite produces constitutively the CymR regulator protein, that binds the Cumate Operator repressing the transcription from the promoter.
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<center><img src="https://static.igem.org/mediawiki/2012/2/2d/TriesteCymR_orange.jpg" width="500px"/></center>
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<h2>Assembly</h2>
<h2>Assembly</h2>
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The CymR gene was obtained by synthesis and then was assembled with other BioBricks.
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<center><img src="https://static.igem.org/mediawiki/2012/b/b0/875003.png" width="300px"/></center>
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<h2>Results</h2>
<h2>Results</h2>
CymR expression was confirmed by Western Blot analysis.  
CymR expression was confirmed by Western Blot analysis.  
Our CymR has a SV5 tag at the C-terminus in order to be detected by an anti-SV5 Ab.
Our CymR has a SV5 tag at the C-terminus in order to be detected by an anti-SV5 Ab.
Its functionality was confirmed through the repression of T5 Cumate Operator-GFP.
Its functionality was confirmed through the repression of T5 Cumate Operator-GFP.
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<img src="https://static.igem.org/mediawiki/2012/d/d0/Trieste-CymR_V.jpg" width="600px"/>
<img src="https://static.igem.org/mediawiki/2012/d/d0/Trieste-CymR_V.jpg" width="600px"/>
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The validation of the repression of the protein CymR on the T5 cumate operator was verified through the creation of a plasmid containing a single copy of CymR  and the T5 cumate operator upstream the GFP (I13504). We saw that the expression of the GFP was completely repressed in absence of p-cumate but it was active in presence of p-cumate.
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<center><img src="https://static.igem.org/mediawiki/igem.org/4/4c/TriesteT5_Cumate_operator_V.jpg" width="500px"/></center>
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<div class="notebook_section">
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    <h2 class="notebook_title">Post European-Jamboree</h2>
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<strong>Cuminaldehyde activation of cumate switch</strong><br/><br/>
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We also tested the activity of the cuminaldehyde in the regulation of the cumate switch and we confirmed that it is able to activate the cumate switch, although in higher concentrations.<br/><br/>
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<center><img src="https://static.igem.org/mediawiki/2012/0/02/TriesteCuminaldehyde.png" width="300px"/></center>
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<p class="didascalia"><strong>Cuminaldehyde test. </strong>We streaked the <i>E.coli</i> containing the plasmid with J23100-CymR-B0015+T5CumateOperator-I13504(GFP) on LB agar plates at different concentration of cuminaldehyde.</p>
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<h2>Looking forward</h2>
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The next step will be the integration of two copies of CymR in the bacterial genome in order to regulate the toxin genes carried by the plasmid.
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<h2>Modelling</h2>
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<h2>Looking forward</h2>
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<h3><a href="http://partsregistry.org/Part:BBa_K875003"target="_blank">Link to the Registry</a></h3>
<h3><a href="http://partsregistry.org/Part:BBa_K875003"target="_blank">Link to the Registry</a></h3>
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                 <li><a href="https://2012.igem.org/Team:Trieste/parts/1">BBa_K875001 - Cumate Op</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/1">BBa_K875001 - Cumate Op</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/2">BBa_K875002 - Lac OP</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/2">BBa_K875002 - Lac OP</a></li>
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                 <li><a href="https://2012.igem.org/Team:Trieste/parts/3">BBa_K875003 - CymR</a></li>
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                 <li class="select" ><a href="https://2012.igem.org/Team:Trieste/parts/3">BBa_K875003 - CymR</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/4">BBa_K875004 - OmpA scFv</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/4">BBa_K875004 - OmpA scFv</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/5">BBa_K875005 - OmpA SIP</a></li>
                 <li><a href="https://2012.igem.org/Team:Trieste/parts/5">BBa_K875005 - OmpA SIP</a></li>

Latest revision as of 18:24, 26 October 2012

BBa_K875003

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Description

This composite produces constitutively the CymR regulator protein, that binds the Cumate Operator repressing the transcription from the promoter.


Assembly

The CymR gene was obtained by synthesis and then was assembled with other BioBricks.



Results

CymR expression was confirmed by Western Blot analysis. Our CymR has a SV5 tag at the C-terminus in order to be detected by an anti-SV5 Ab. Its functionality was confirmed through the repression of T5 Cumate Operator-GFP.

The validation of the repression of the protein CymR on the T5 cumate operator was verified through the creation of a plasmid containing a single copy of CymR and the T5 cumate operator upstream the GFP (I13504). We saw that the expression of the GFP was completely repressed in absence of p-cumate but it was active in presence of p-cumate.



Post European-Jamboree

Cuminaldehyde activation of cumate switch

We also tested the activity of the cuminaldehyde in the regulation of the cumate switch and we confirmed that it is able to activate the cumate switch, although in higher concentrations.



Cuminaldehyde test. We streaked the E.coli containing the plasmid with J23100-CymR-B0015+T5CumateOperator-I13504(GFP) on LB agar plates at different concentration of cuminaldehyde.





Looking forward

The next step will be the integration of two copies of CymR in the bacterial genome in order to regulate the toxin genes carried by the plasmid.


Link to the Registry


Università degli studi di Trieste ICGEB Illy Fondazione Cassa di Risparmio
iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012
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