Team:Trieste/parts/3

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     <h2 class="notebook_title">Post European-Jamboree</h2>
     <h2 class="notebook_title">Post European-Jamboree</h2>
<strong>Cuminaldehyde activation of cumate switch</strong><br/><br/>
<strong>Cuminaldehyde activation of cumate switch</strong><br/><br/>
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Up to now all our tests were based on p-cumate according  to the literature <p>(Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains
Young J. Choi, Lyne Morel, Teffanie Le François Denis Bourque, Lucie Bourget, Denis Groleau, Bernard Massie and Carlos B. Míguez
Applied and Environmental Microbiology, Aug. 2010, p. 5058–5066 Vol.76 doi:10.1128/AEM.00413-10)</p>.
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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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But since it is known that the most abundant component in the cumin spice is the cuminaldehyde,  we  later decided to test also its activity in the regulation of the cumate switch. We confirmed that also the cuminaldehyde was 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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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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