Team:Trieste/parts/1

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                 <div class="box_contenuti">
<h2>Description </h2>  
<h2>Description </h2>  
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</br>  
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This promoter is composed by phage T5 promoter and Cumate operator. It is repressed in the  presence of CymR protein which binds the Cumate Operator.
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<br/>
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</br>
<h2>Assembly</h2>
<h2>Assembly</h2>
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</br>  
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Obtained by synthesis
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</br>
 +
</br>
<h2>Results</h2>
<h2>Results</h2>
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</br>  
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To test this promoter we performed two different assays.
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First of all we cloned this promoter in pSB1C3 and then we inserted downstream the GFP BBa_I13504. In the same plasmid we cloned J23100-CymR-B0015 in order to repress the promoter. To test this system we used different concentrations of cumate which binds CymR preventing its repression, thus allowing the GFP expression.
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</br>
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</br>
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<b>In liquid assay:</b>
 +
</br>
 +
</br>
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We inoculated a 20ml culture. After overnight growth, we diluted the culture to OD600 = 0.2. Then we aliquoted 200 μl in 8 replicates in a microtiter plate at different concentrations of cumate. The reading was performed in a monochromator at 485-510nm.
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</br>
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<center><img src="https://static.igem.org/mediawiki/igem.org/1/18/Trieste_liquid_assay.png" width="450px"/><br /></center>
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</br>
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</br>
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<b>In the plate assay:</b>
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</br>
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</br>
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We streaked the culture on LB plates containing  different cumate concentrations
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</br>
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</br>
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<center><img src="https://static.igem.org/mediawiki/igem.org/0/0c/T5_Cumate_operator_V.jpg" width="600px"/></br>
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</br>
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</br>
<h2>Modelling</h2>
<h2>Modelling</h2>
</br>  
</br>  

Revision as of 10:43, 25 September 2012

BBa_K875001

More

Description

This promoter is composed by phage T5 promoter and Cumate operator. It is repressed in the presence of CymR protein which binds the Cumate Operator.

Assembly

Obtained by synthesis

Results

To test this promoter we performed two different assays. First of all we cloned this promoter in pSB1C3 and then we inserted downstream the GFP BBa_I13504. In the same plasmid we cloned J23100-CymR-B0015 in order to repress the promoter. To test this system we used different concentrations of cumate which binds CymR preventing its repression, thus allowing the GFP expression.

In liquid assay:

We inoculated a 20ml culture. After overnight growth, we diluted the culture to OD600 = 0.2. Then we aliquoted 200 μl in 8 replicates in a microtiter plate at different concentrations of cumate. The reading was performed in a monochromator at 485-510nm.



In the plate assay:

We streaked the culture on LB plates containing different cumate concentrations




Modelling


Looking forward


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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