Team:UCL London/Research/Supercoilometer/Experiments

From 2011.igem.org

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<h1>Building a Supercoilometer in the lab</h1>
<h1>Building a Supercoilometer in the lab</h1>
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The first step to construct the device was to clone the tyrT promoter from the E coli genome by performing PCR. The relevant primers (with necessary prefix and suffix for BioBrick standardisation) was designed and synthesised in order to clone out the promoter sequence of interest. Following this a 3A assembly was carried out using the cloned tyrT promoter, a CFP expression cassette (from iGEM 2011 Kit Plate 1 - 8M) and standard plasmid backbone (pSB1C3) to construct the final device.
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<h2>Aim</h2>
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To prepare a BioBrick from the tyrT promoter cloned from the E. coli genome and then construct and characterise a device which can detect the level of negative supercoiling in a closed circular plasmid.
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For characterisation, the device was further ligated to a plasmid backbone containing the Mu gyrase binding site. Two shake flask cultures were carried out, one with the device on a normal plasmid and another with device ligated to the Mu GBS. Cell samples were collected from both flasks every 2 hours for a duration of 24 hours, and the CFP fluorescence level was measured to detect a difference. The remaining cell sample at the end of the culture would be mini-prepped and run on a chloroquine gel to look at the difference in the level of supercoiling density.  Further work will be needed to carry
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<h2>Parts used</h2>
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<html><table border="0" cellspacing="0" cellpadding="0">
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  <tr>
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    <td width="91" valign="top" bgcolor="#CCCCCC"><p><strong>Part Name</strong></p></td>
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    <td width="82" valign="top" bgcolor="#CCCCCC"><p align="center"><strong>Parts  ID</strong></p></td>
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    <td width="146" valign="top" bgcolor="#CCCCCC"><p align="center"><strong>Parts Description</strong></p></td>
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    <td width="90" valign="top" bgcolor="#CCCCCC"><p align="center"><strong>Length (bp)</strong></p></td>
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    <td width="150" valign="top" bgcolor="#CCCCCC"><p align="center"><strong>Plasmid Backbone</strong></p></td>
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  <tr>
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    <td width="91" valign="top"><p>RBS+CFP.LVA+Terminator</p></td>
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    <td width="82" valign="top"><p><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032">BBa_E0422</a></p></td>
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    <td width="146" valign="top"><p>RBS + cyan fluorescent protein with LVA tag +  terminator</p></td>
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    <td width="53" valign="top"><p align="center">917</p></td>
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    <td width="109" valign="top"><p>on pSB1A2 plasmid (2079 bp)  - ampicillin resistance</p></td>
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  </tr>
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  <tr>
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    <td width="91" valign="top"><p><a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a></p></td>
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    <td width="82" valign="top"><p align="center">n/a</p></td>
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    <td width="146" valign="top"><p>standard plasmid backbone (chloramphenicol  resistance)</p></td>
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    <td width="53" valign="top"><p align="center">2070</p></td>
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    <td width="109" valign="top"><p>n/a</p></td>
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  </tr>
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</table></html>
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Revision as of 23:27, 21 September 2011

Building a Supercoilometer in the lab

Aim

To prepare a BioBrick from the tyrT promoter cloned from the E. coli genome and then construct and characterise a device which can detect the level of negative supercoiling in a closed circular plasmid.

Parts used

Part Name

Parts  ID

Parts Description

Length (bp)

Plasmid Backbone

RBS+CFP.LVA+Terminator

BBa_E0422

RBS + cyan fluorescent protein with LVA tag + terminator

917

on pSB1A2 plasmid (2079 bp) - ampicillin resistance

pSB1C3

n/a

standard plasmid backbone (chloramphenicol resistance)

2070

n/a

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