http://2007.igem.org/wiki/index.php?title=Special:Contributions/LucasCY&feed=atom&limit=50&target=LucasCY&year=&month=2007.igem.org - User contributions [en]2024-03-28T14:00:01ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/ImperialImperial2007-10-27T03:34:57Z<p>LucasCY: </p>
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<h2><b>Welcome!</b></h2><br />
<div class="contentbox">The IC GEMs team consists of ten 2nd year undergraduates from Bioengineering and Bioscience. Our genuine collective interest in healthcare led to the targetting of <b>health and medicine related applications</b> this year. Together with the vision of an industry, the strategy we adopted for our projects is to use the Engineering Cycle to create reliable Synthetic Biology products where standards and quality control are strictly kept.<br />
<br />
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<h1><b>Main Project</b> - <a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction">Infector Detector</a></h1> <br />
<br />
Infector Detector is a system that will detect the presence of biofilm infections on urinary catheters by reporting on the presence of AHL, a signalling molecule used by E.<i>coli</i>, and output a fluorescent protein as a result. (<a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction">more...</a>)<br />
<br><br><br />
<br />
<table style="background-color: #f5facf;" width="590px" align="center" border="0" cellpadding="7px" cellspacing="5"><br />
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<td valign="top" style="text-align:justify;"><br />
<center><img src="https://static.igem.org/mediawiki/2007/d/d5/IC07_IDnews.png" width="260px"></center><br />
</td><br />
<td><br />
<div id="SummaryBox"><br />
<h2>Main Achievements</h2><br />
<div class="parabox"><br />
<ul><br />
<li>Designed and modelled a device to <b>detect biofilms</b> and <b>prevent urinary catheter infections</b> like MRSA<br />
<li>Successfully utilized <b>cell-free chassis</b> for Infector Detector<br />
<li>Characterised Infector Detector to <b>detect AHL at ambient temperatures</b><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison"><b>Compared system expression</b></a> to <a href="http://partsregistry.org/Part:BBa_F2620">AHL Receiver (BBa_F2620)</a> transfer function.<br />
</ul><br />
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<h2><b>Foundational technology</b> - <a href="https://2007.igem.org/Imperial/Cell-Free/Whatis">Cell-Free Systems</a></h2><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2007/b/bb/IC07_CFS_components.png" width="400px"></center><br />
<table style="background-color: #dff7d6;" width="560px" align="center" border="0" cellpadding="7px" cellspacing="5"><br />
<td valign="top" style="text-align:justify;">Power your iGEM projects with Cell-Free Systems! Find out what this new chassis has done for us, and what it can do for you! (<a href="https://2007.igem.org/Imperial/Cell-Free/Whatis">more...</a>)<br><br />
<br />
</table><br />
</div><br />
</div> <br />
<br />
<br />
<div id="FeatureBox"><br />
<div class="contentbox"><br />
<h1><b>Sub Project</b> - <a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction">Cell by Date</a></h1> <br />
<br />
Cell by Date will report when ground beef has been out of the cold chain for too long. It produces fluorescent proteins at ambient temperatures above 8&deg;C. (<a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction">more...</a>)<br><br />
<br />
<table style="background-color: #f5facf;" width="560px" align="center" border="0" cellpadding="7px" cellspacing="5"><br />
<tr><br />
<td valign="center"><img src="https://static.igem.org/mediawiki/2007/7/74/CBDCartoonfront.png" width="220px"><br />
<br />
<td valign="top" style="text-align:left;"><br />
<div id="SummaryBox" style="width: 300px;"><br />
<h2>Main Achievements</h2><br />
<div class="parabox" style="width: 270px;"><br />
<ul><br />
<li>Designed and modelled a device to <b>indicate spoilage of meat</b>/break in the cold chain via the <b>thermal history</b> of the system<br />
<li> Utilised project results to <b>characterise cell-free chassis</b><br />
<li> Characterised <a href="http://partsregistry.org/Part:BBa_I13522">pTet-GFP</a> and <a href="http://partsregistry.org/Part:BBa_I719015">pT7-GFP</a> under <b>isothermal conditions</b><br />
</ul><br />
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<h2><b><a href="https://2007.igem.org/Imperial/Team" title="">Meet the Team</a></b></h2><br />
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<img src="https://static.igem.org/mediawiki/2007/9/97/IC07_teamphoto3.jpg" width="230px"><br />
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<h2><b> <a href="https://2007.igem.org/Imperial/Open_Science_on_OpenWetWare" title="">Open Science on OpenWetWare</a></b></h2><br />
<table style="background-color: #d9f0f8; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<tr style="font-weight: bold;"><br />
<td>We did Open Science!</td><br />
</tr><tr><br />
<td><br />
Our projects were run and organised using OWW, with updates on a day-to-day basis! Click <a href="https://2007.igem.org/Imperial/Open_Science_on_OpenWetWare" title="">here to access our <b>400+ pages of full documentation!</b></a><br />
</td></tr><br />
</table><br />
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<br />
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<h2><b>Contributions to Registry</b></h2><br />
<table style="background-color: #f0dbcc; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<tr style="font-weight: bold;"><br />
<td>Chassis Additions</td><br />
</tr><tr><br />
<td><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems">Cell-Free Systems</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Homemade_E.coli_S30">- Homemade <i>E. coli</i> S30</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Commercial_E.coli_S30">- Commercial <i>E. coli</i> S30</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Commercial_E.coli_T7_S30">- Commercial <i>E. coli</i> T7 S30</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Vesicle">- Vesicle Encapsulation</a><br><br />
</td></tr><br />
<tr style="font-weight: bold;"><br />
<td>Part/Construct Additions</td><br />
</tr><tr><br />
<td><br />
<a href="http://partsregistry.org/Part:BBa_I719005">T7 promoter (BBa_I719005)</a><br><br />
<a href="http://partsregistry.org/Part:BBa_I719015">pT7 with GFP reporter (BBa_I719015)</a><br />
</td></tr><br />
<tr style="font-weight: bold;"><br />
<td>Part/Construct Characterisation</td><br />
</tr><tr><br />
<td><br />
<a href="http://partsregistry.org/Part:BBa_F2620"> AHL Receiver (BBa_F2620)</a> <br><br />
<a href="http://partsregistry.org/Part:BBa_I13522">pTet GFP (BBa_I13522)</a><br><br />
<a href="http://partsregistry.org/Part:BBa_J37032">pLux GFP (BBa_J37032)</a><br><br />
<br />
</td><br />
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<h2><b><a href="https://2007.igem.org/Imperial/Fun" title="">Fun with iGEM</a></b></h2><br />
<table style="background-color: #f7fbd7; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<td> Pictures, Quotes and More!<br />
</table><br />
<br />
</div><br />
<br />
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<h2><b><a href="https://2007.igem.org/Imperial/Acknowledgements" title="">Acknowledgements</a></b></h2><br />
<table style="background-color: #eeeeee; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<td><br />
IC Deputy Rector's Fund<br><br />
IC Faculty of Engineering<br><br />
IC Faculty of Natural Sciences<br><br />
European Commission, Synbiocomm Project<br><br />
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</html></div>LucasCYhttp://2007.igem.org/wiki/index.php/ImperialImperial2007-10-27T03:31:22Z<p>LucasCY: </p>
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<br />
<!-- Start of Central Content Box --><br />
<br />
<div id="maincol"><br />
<br />
<br />
<div id="OverviewBox"><br />
<h2><b>Welcome!</b></h2><br />
<div class="contentbox">The IC GEMs team consists of ten 2nd year undergraduates from Bioengineering and Bioscience. Our genuine collective interest in healthcare led to the targetting of <b>health and medicine related applications</b> this year. Together with the vision of an industry, the strategy we adopted for our projects is to use the Engineering Cycle to create reliable Synthetic Biology products where standards and quality control are strictly kept.<br />
<br />
</div><br />
</div><br />
<br />
<div id="FeatureBox"><br />
<div class="contentbox"><br />
<h1><b>Main Project</b> - <a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction">Infector Detector</a></h1> <br />
<br />
Infector Detector is a system that will detect the presence of biofilm infections on urinary catheters by reporting on the presence of AHL, a signalling molecule used by E.<i>coli</i>, and output a fluorescent protein as a result. (<a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction">more...</a>)<br />
<br><br><br />
<br />
<table style="background-color: #f5facf;" width="590px" align="center" border="0" cellpadding="7px" cellspacing="5"><br />
<tr><br />
<td valign="top" style="text-align:justify;"><br />
<center><img src="https://static.igem.org/mediawiki/2007/d/d5/IC07_IDnews.png" width="260px"></center><br />
</td><br />
<td><br />
<div id="SummaryBox"><br />
<h2>Main Achievements</h2><br />
<div class="parabox"><br />
<ul><br />
<li>Designed and modelled a device to <b>detect biofilms</b> and <b>prevent urinary catheter infections</b> like MRSA<br />
<li>Successfully utilized <b>cell-free chassis</b> for Infector Detector<br />
<li>Characterised Infector Detector to <b>detect AHL at ambient temperatures</b><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison"><b>Compared system expression</b></a> to <a href="http://partsregistry.org/Part:BBa_F2620">AHL Receiver (BBa_F2620)</a> transfer function.<br />
</ul><br />
</div><br />
</div><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div> <br />
<br />
<br />
<div id="TechnoBox"><br />
<div class="contentbox"><br />
<h2><b>Foundational technology</b> - <a href="https://2007.igem.org/Imperial/Cell-Free/Whatis">Cell-Free Systems</a></h2><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2007/b/bb/IC07_CFS_components.png" width="400px"></center><br />
<table style="background-color: #dff7d6;" width="560px" align="center" border="0" cellpadding="7px" cellspacing="5"><br />
<td valign="top" style="text-align:justify;">Power your iGEM projects with Cell-Free Systems! Find out what this new chassis has done for us, and what it can do for you! (<a href="https://2007.igem.org/Imperial/Cell-Free/Whatis">more...</a>)<br><br />
<br />
</table><br />
</div><br />
</div> <br />
<br />
<br />
<div id="FeatureBox"><br />
<div class="contentbox"><br />
<h1><b>Sub Project</b> - <a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction">Cell by Date</a></h1> <br />
<br />
Cell by Date will report when ground beef has been out of the cold chain for too long. It produces fluorescent proteins at ambient temperatures above 8&deg;C. (<a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction">more...</a>)<br><br />
<br />
<table style="background-color: #f5facf;" width="560px" align="center" border="0" cellpadding="7px" cellspacing="5"><br />
<tr><br />
<td valign="center"><img src="https://static.igem.org/mediawiki/2007/7/74/CBDCartoonfront.png" width="200px"><br />
<br />
<td valign="top" style="text-align:left;"><br />
<div id="SummaryBox" style="width: 300px;"><br />
<h2>Main Achievements</h2><br />
<div class="parabox" style="width: 270px;"><br />
<ul><br />
<li>Designed and modelled a device to <b>indicate spoilage of meat</b>/break in the cold chain via the <b>thermal history</b> of the system<br />
<li> Utilised project results to <b>characterise cell-free chassis</b><br />
<li> Characterised <a href="http://partsregistry.org/Part:BBa_I13522">pTet-GFP</a> and <a href="http://partsregistry.org/Part:BBa_I719015">pT7-GFP</a> under <b>isothermal conditions</b><br />
</ul><br />
</div><br />
</div><br />
</td><br />
</tr><br />
</table><br />
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<h2><b><a href="https://2007.igem.org/Imperial/Team" title="">Meet the Team</a></b></h2><br />
<br /><br />
<img src="https://static.igem.org/mediawiki/2007/9/97/IC07_teamphoto3.jpg" width="230px"><br />
<br><br />
</div><br />
<br />
<div class="post-it bluebox"><br />
<h2><b> <a href="https://2007.igem.org/Imperial/Open_Science_on_OpenWetWare" title="">Open Science on OpenWetWare</a></b></h2><br />
<table style="background-color: #d9f0f8; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<tr style="font-weight: bold;"><br />
<td>We did Open Science!</td><br />
</tr><tr><br />
<td><br />
Our projects were run and organised using OWW, with updates on a day-to-day basis! Click <a href="https://2007.igem.org/Imperial/Open_Science_on_OpenWetWare" title="">here to access our <b>400+ pages of full documentation!</b></a><br />
</td></tr><br />
</table><br />
</div><br />
<br />
<div class="post-it pinkbox"><br />
<h2><b>Contributions to Registry</b></h2><br />
<table style="background-color: #f0dbcc; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<tr style="font-weight: bold;"><br />
<td>Chassis Additions</td><br />
</tr><tr><br />
<td><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems">Cell-Free Systems</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Homemade_E.coli_S30">- Homemade <i>E. coli</i> S30</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Commercial_E.coli_S30">- Commercial <i>E. coli</i> S30</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Commercial_E.coli_T7_S30">- Commercial <i>E. coli</i> T7 S30</a><br><br />
<a href="http://partsregistry.org/Chassis/Cell-Free_Systems/Vesicle">- Vesicle Encapsulation</a><br><br />
</td></tr><br />
<tr style="font-weight: bold;"><br />
<td>Part/Construct Additions</td><br />
</tr><tr><br />
<td><br />
<a href="http://partsregistry.org/Part:BBa_I719005">T7 promoter (BBa_I719005)</a><br><br />
<a href="http://partsregistry.org/Part:BBa_I719015">pT7 with GFP reporter (BBa_I719015)</a><br />
</td></tr><br />
<tr style="font-weight: bold;"><br />
<td>Part/Construct Characterisation</td><br />
</tr><tr><br />
<td><br />
<a href="http://partsregistry.org/Part:BBa_F2620"> AHL Receiver (BBa_F2620)</a> <br><br />
<a href="http://partsregistry.org/Part:BBa_I13522">pTet GFP (BBa_I13522)</a><br><br />
<a href="http://partsregistry.org/Part:BBa_J37032">pLux GFP (BBa_J37032)</a><br><br />
<br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="post-it yellowbox"><br />
<h2><b><a href="https://2007.igem.org/Imperial/Fun" title="">Fun with iGEM</a></b></h2><br />
<table style="background-color: #f7fbd7; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<td> Pictures, Quotes and More!<br />
</table><br />
<br />
</div><br />
<br />
<div class="post-it whitebox" style="filter:alpha(opacity=80); -moz-opacity:0.8;"><br />
<h2><b><a href="https://2007.igem.org/Imperial/Acknowledgements" title="">Acknowledgements</a></b></h2><br />
<table style="background-color: #eeeeee; filter:alpha(opacity=80); -moz-opacity:0.8; font-size: .95em;"><br />
<td><br />
IC Deputy Rector's Fund<br><br />
IC Faculty of Engineering<br><br />
IC Faculty of Natural Sciences<br><br />
European Commission, Synbiocomm Project<br><br />
</td><br />
</table><br />
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</html></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-27T03:26:38Z<p>LucasCY: </p>
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<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
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__NOTOC__<br />
=Infector Detector: Conclusion =<br />
The main achievements of the Infector Detector project:<br />
*Extensive modelling of the two potential constructs for Infector Detector<br />
*Purification of GFPmut3b to allow construction of a calibration curve <br />
*Detailed characterisation of construct 1 ''in vitro'' using a calibration curve to find rate of GFP synthesis<br />
*Creation of a standard unit to allow comparison between ''in vitro'' and ''in vivo''<br />
<br />
The table below summarises our Infector Detector system in the context of the original specifications:<br />
<br />
{|border="1" width="80%" align="center"<br />
|-<br />
|width="20%" style="background:#ffffcc"|<center>'''Property'''</center><br />
|width="--"|<center>'''Specification'''</center><br />
|'''Achievements'''<br />
|-<br />
|style="background:#ffffcc"|Inputs<br />
|<center>System must be sensitive to AHL concentration between 5-50nM</center><br />
|'''<font color=green>Sensitive to 5-1000nM</font>'''<br />
|-<br />
|style="background:#ffffcc"|Outputs<br />
|<center>System must give a visual signal if bacteria is present</center><br />
|'''<font color=red>To Be Determined - Using Stronger fluorescent protein such as DsRed express</font>''' <br />
|-<br />
|style="background:#ffffcc"|Response Time<br />
|<center>System needs to have a response time under 3 hours</center><br />
|'''<font color=green>Systems responds <30minutes and reaches peak fluorescence at 300minutes'''</font><br />
|-<br />
|style="background:#ffffcc"|Operating Conditions<br />
|<center>System must operate within temperature 20-30&deg;C</center><br />
|'''<font color=green>System works at 25&deg;C</font>'''<br />
|-<br />
|style="background:#ffffcc"|Health & Safety<br />
|<center>System Must not be living, replicating bacteria, and in any way harmful or infectious.</center><br />
|'''<font color=green>Cell Free ''in vitro'' chassis</font>'''<br />
|-<br />
|style="background:#ffffcc"|Shelf-life<br />
|<center>System must have a shelf life of 7 days</center><br />
|'''<font color=green>Can be stored in freezer for prolonged periods</font>'''<br />
|-<br />
|style="background:#ffffcc"|Packaging and Application<br />
|<center>System must be portable and convenient to use</center><br />
|'''<font color=red>To Be Determined- Using our chassis in a spray</font>''' <br />
|}<br />
<br clear="all"><br />
<br />
Infector detector utilises synthetic biology to fight one of the most common infections rampaging through hospitals worldwide. Urinary catheter infections and catheter-related bacteremias in general have been troubling both doctors and patients for years now on since the infection is usually very difficult to detect at an early stage. Infector detector battles the infection with its own weapons by utilising its quorum sensing mechanism to initiate a fluorescence reporter, signaling the presence of a biofilm in the making. Upon noticing the change in colour, medical staff will replace the catheter before the infection spreads. Thus doctors can rest assured that their patients get the protection they need. And all this without invoking a single bacterium, thanks to our Cell-free chassis.<br />
<br />
Its evolution cannot however stop here ! While charactrising infector detector we came across certain aspects that can be further developed to enhance its functions. These are described in more detail further below:<br />
<br />
<br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector Detector lies in that it isn't limited to just one type of infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By using different homologues to the LuxR quorum sensing, Infector Detector can be used to battle a range of catheter-related bacteria. For example by using, a construct that recognises AI-2<sup>[[#References |1]]</sup> we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:IC2007 Conculsion4.jpg|thumb|right|390px|Tweaking sensitivity using LuxR]] <br />
The main advantages of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity. In addition by adding purified LuxR the chassis does not have to produce LuxR and so has more energy to produce GFP.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity.<br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged as either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as they can simply spray from a distance. The disadvantage is the poor accuracy of application, waste, and higher rate of evaporation.<br />
<br />
<br />
A cream, on the other hand, will decrease significantly any evaporation and will allow the user to apply the detector to specific areas of the catheter with more control. The disadvantage is that the diffusion rate of AHL and detection compounds through a viscous cream is lower. This will slow down system response.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
As you can see, the development of the application can easily be extended into many areas, and even though it is far from being commercially available, Infector Detector has proven that even the tiniest of members of the microcosm around us can tackle a problem of worldwide dimensions.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison << F2620 Comparison] | Conclusions | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-27T03:16:04Z<p>LucasCY: /* Packaging */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<br clear="all"><br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
=Infector Detector: Conclusion =<br />
The main achievements of the Infector Detector project:<br />
*Extensive modelling of the two potential constructs for Infector Detector<br />
*Purification of GFPmut3b to allow construction of a calibration curve <br />
*Detailed characterisation of construct 1 ''in vitro'' using a calibration curve to find rate of GFP synthesis<br />
*Creation of a standard unit to allow comparison between ''in vitro'' and ''in vivo''<br />
<br />
The table below summarises our Infector Detector system in the context of the original specifications:<br />
<br />
{|border="1" width="80%" align="center"<br />
|-<br />
|width="20%" style="background:#ffffcc"|<center>'''Property'''</center><br />
|width="--"|<center>'''Specification'''</center><br />
|'''Achievements'''<br />
|-<br />
|style="background:#ffffcc"|Inputs<br />
|<center>System must be sensitive to AHL concentration between 5-50nM</center><br />
|'''<font color=green>Sensitive to 5-1000nM</font>'''<br />
|-<br />
|style="background:#ffffcc"|Outputs<br />
|<center>System must give a visual signal if bacteria is present</center><br />
|'''<font color=red>To Be Determined - Using Stronger fluorescent protein such as DsRed express</font>''' <br />
|-<br />
|style="background:#ffffcc"|Response Time<br />
|<center>System needs to have a response time under 3 hours</center><br />
|'''<font color=green>Systems responds <30minutes and reaches peak fluorescence at 300minutes'''</font><br />
|-<br />
|style="background:#ffffcc"|Operating Conditions<br />
|<center>System must operate within temperature 20-30&deg;C</center><br />
|'''<font color=green>System works at 25&deg;C</font>'''<br />
|-<br />
|style="background:#ffffcc"|Health & Safety<br />
|<center>System Must not be living, replicating bacteria, and in any way harmful or infectious.</center><br />
|'''<font color=green>Cell Free ''in vitro'' chassis</font>'''<br />
|-<br />
|style="background:#ffffcc"|Shelf-life<br />
|<center>System must have a shelf life of 7 days</center><br />
|'''<font color=green>Can be stored in freezer for prolonged periods</font>'''<br />
|-<br />
|style="background:#ffffcc"|Packaging and Application<br />
|<center>System must be portable and convenient to use</center><br />
|'''<font color=red>To Be Determined- Using our chassis in a spray</font>''' <br />
|}<br />
<br clear="all"><br />
<br />
Infector detector utilises synthetic biology to fight one of the most common infections rampaging through hospitals worldwide. Urinary catheter infections and catheter-related bacteria in general have been troubling both doctors and patients for years now on since the infection is usually very difficult to detect at an early stage. Infector detector battles the infection with its own weapons by utilising its quorum sensing mechanism to initiate a fluorescence reporter, signaling the presence of a biofilm in the making. Upon noticing the change in colour, medical staff will replace the catheter before the infection spreads. Thus doctors can rest assured that their patients get the protection they need. And all this without invoking a single bacterium, thanks to our Cell-free chassis.<br />
<br />
Its evolution cannot however stop here ! While charactrising infector detector we came across certain aspects that can be further developed to enhance its functions. These are described in more detail further below:<br />
<br />
<br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector Detector lies in that it isn't limited to just one type of infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By using different homologues to the LuxR quorum sensing, Infector Detector can be used to battle a range of catheter-related bacterium. For example by using, a construct that recognises AI-2<sup>[[#References |1]]</sup> we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:IC2007 Conculsion4.jpg|thumb|right|390px|Tweaking sensitivity using LuxR]] <br />
The main advantages of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity. In addition by adding purified LuxR the chassis does not have to produce LuxR and so has more energy to produce GFP.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity.<br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged as either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as they can simply spray from a distance. The disadvantage is the poor accuracy of application, waste, and higher rate of evaporation.<br />
<br />
<br />
A cream, on the other hand, will decrease significantly any evaporation and will allow the user to apply the detector to specific areas of the catheter with more control. The disadvantage is that the diffusion rate of AHL and detection compounds through a viscous cream is lower. This will slow down system response.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
As you can see, the development of the application can easily be extended into many areas, and even though it is far from being commercially available, Infector Detector has proven that even the tiniest of members of the microcosm around us can tackle a problem of worldwide dimensions.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison << F2620 Comparison] | Conclusions | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-27T01:31:42Z<p>LucasCY: /* Infector Detector: Conclusion */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
=Infector Detector: Conclusion =<br />
The main achievements of the Infector Detector project:<br />
*Extensive modelling of the two potential constructs for Infector Detector<br />
*Purification of GFPmut3b to allow construction of a calibration curve <br />
*Detailed characterisation of construct 1 ''in vitro'' using a calibration curve to find rate of GFP synthesis<br />
*Creation of a standard unit to allow comparison between ''in vitro'' and ''in vivo''<br />
<br />
The table below summarises our Infector Detector system in the context of the original specifications:<br />
<br />
{|border="1" width="80%" align="center"<br />
|-<br />
|width="20%" style="background:#ffffcc"|<center>'''Property'''</center><br />
|width="--"|<center>'''Specification'''</center><br />
|'''Achievements'''<br />
|-<br />
|style="background:#ffffcc"|Inputs<br />
|<center>System must be sensitive to AHL concentration between 5-50nM</center><br />
|'''<font color=green>Sensitive to 5-1000nM</font>'''<br />
|-<br />
|style="background:#ffffcc"|Outputs<br />
|<center>System must give a visual signal if bacteria is present</center><br />
|'''<font color=red>Future work - Using Stronger fluorescent protein such as DsRed express</font>''' <br />
|-<br />
|style="background:#ffffcc"|Response Time<br />
|<center>System needs to have a response time under 3 hours</center><br />
|'''<font color=green>Systems responds <30minutes</font>'''<br />
|-<br />
|style="background:#ffffcc"|Operating Conditions<br />
|<center>System must operate within temperature 20-30&deg;C</center><br />
|'''<font color=green>System works at 25&deg;C</font>'''<br />
|-<br />
|style="background:#ffffcc"|Health & Safety<br />
|<center>System Must not be living, replicating bacteria, and in any way harmful or infectious.</center><br />
|'''<font color=green>Cell Free ''in vitro'' chassis</font>'''<br />
|-<br />
|style="background:#ffffcc"|Shelf-life<br />
|<center>System must have a shelf life of 7 days</center><br />
|'''<font color=green>Can be stored in freezer for prolonged periods</font>'''<br />
|-<br />
|style="background:#ffffcc"|Packaging<br />
|<center>System must be portable and convenient to use</center><br />
|'''<font color=red>Future Work - Using our chassis in a spray</font>''' <br />
|}<br />
<br clear="all"><br />
<br />
Infector detector utilises synthetic biology to fight one of the most common infections rampaging through hospitals worldwide. Urinary catheter infections and catheter-related bacteremias in general have been troubling both doctors and patients for years now on since the infection is usually very difficult to detect at an early stage. Infector detector battles the infection with its own weapons by utilising its quorum sensing mechanism to initiate a fluorescence reporter, signalling the presence of a biofilm in the making. Upon noticing the change in colour, medical staff will replace the catheter before the infection spreads. Thus doctors can rest assured that their patients get the protection they need. And all this without invoking a single bacterium, thanks to our Cell-free chasis.<br />
<br />
Its evolution cannot however stop here ! While charactrising infector detector we came across certain aspects that can be further developed to enhance its functions. These are described in more detail further below:<br />
<br />
<br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector Detector lies in that it isn't limited to just one type of infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. By using.for example, a construct that recognises AI-2<sup>[[#References |1]]</sup> we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:IC2007 Conculsion4.jpg|thumb|right|390px|Tweaking sensitivity using LuxR]] <br />
The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br />
<br />
<br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity.<br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged as either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as they can simply spray from a distance. The disadvantage is the poor accuracy of application, waste, and higher rate of evaporation.<br />
<br />
<br />
A cream, on the other hand, will decrease significantly any evaporation and will allow the user to apply the detector to specific areas of the catheter with more control. The disadvantage is that the diffusion rate of AHL and detection compounds through a viscous cream is lower. This will slow down system repsonse.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
So as you can see, the development of this application can easily be extended into many areas, and even though it is far from being commercially available, it has proven that even the tiniest bits of the microcosm around us can be used to tackle a worldwide problem.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison << F2620 Comparison] | Conclusions | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-27T01:23:43Z<p>LucasCY: </p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
=Infector Detector: Conclusion =<br />
The main achievements of the Infector Detector project:<br />
*Extensive modelling of the two potential constructs for Infector Detector<br />
*Purification of GFPmut3b to allow construction of a calibration curve <br />
*Detailed characterisation of construct 1 ''in vitro'' using a calibration curve to find rate of GFP synthesis<br />
*Creation of a standard unit to allow comparison between ''in vitro'' and ''in vivo''<br />
<br />
The table below summarises our Infector Detector system in the context of the original specifications:<br />
<br />
{|border="1" width="80%" align="center"<br />
|-<br />
|width="20%" style="background:#ffffcc"|<center>'''Property'''</center><br />
|width="--"|<center>'''Specification'''</center><br />
|'''Achievements'''<br />
|-<br />
|style="background:#ffffcc"|Inputs<br />
|<center>System must be sensitive to AHL concentration between 5-50nM</center><br />
|'''<font color=green>Sensitive to 5-1000nM</font>'''<br />
|-<br />
|style="background:#ffffcc"|Outputs<br />
|<center>System must give a visual signal if bacteria is present</center><br />
|'''<font color=red>Future work - Using Stronger fluorescent protein such as DsRed express</font>''' <br />
|-<br />
|style="background:#ffffcc"|Response Time<br />
|<center>System needs to have a response time under 3 hours</center><br />
|'''<font color=green>Systems responds <30minutes</font>'''<br />
|-<br />
|style="background:#ffffcc"|Operating Conditions<br />
|<center>System must operate within temperature 20-30&deg;C</center><br />
|'''<font color=green>System works at 25&deg;C</font>'''<br />
|-<br />
|style="background:#ffffcc"|Health & Safety<br />
|<center>System Must not be living, replicating bacteria, and in any way harmful or infectious.</center><br />
|'''<font color=green>Cell Free ''in vitro'' chassis</font>'''<br />
|-<br />
|style="background:#ffffcc"|Shelf-life<br />
|<center>System must have a shelf life of 7 days</center><br />
|'''<font color=green>Can be stored in freezer for prolonged periods</font>'''<br />
|-<br />
|style="background:#ffffcc"|Packaging<br />
|<center>System must be portable and convenient to use</center><br />
|'''<font color=red>Future Work - Using our chassis in a spray</font>''' <br />
|}<br />
<br clear="all"><br />
<br />
Infector detector utilises synthetic biology to fight one of the most common infections rampaging through hospitals worldwide. Urinary catheter infections and catheter-related bacteremias in general have been troubling both doctors and patients for years now on since the infection is usually very difficult to detect at an early stage. Infector detector battles the infection with its own weapons by utilising its quorum sensing mechanism to initiate a fluorescence reporter, signalling the presence of a biofilm in the making. Upon noticing the change in colour, medical staff will replace the catheter before the infection spreads. Thus doctors can rest assured that their patients get the protection they need. And all this without invoking a single bacterium, thanks to our Cell-free chasis.<br />
<br />
Its evolution cannot however stop here ! While charactrising infector detector we came across certain aspects that can be further developed to enhance its functions. These are described in more detail further below.<br />
<br />
<br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector Detector lies in that it isn't limited to just one type of infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. By using.for example, a construct that recognises AI-2<sup>[[#References |1]]</sup> we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:IC2007 Conculsion4.jpg|thumb|right|390px|Tweaking sensitivity using LuxR]] <br />
The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br />
<br />
<br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity.<br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged as either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as they can simply spray from a distance. The disadvantage is the poor accuracy of application, waste, and higher rate of evaporation.<br />
<br />
<br />
A cream, on the other hand, will decrease significantly any evaporation and will allow the user to apply the detector to specific areas of the catheter with more control. The disadvantage is that the diffusion rate of AHL and detection compounds through a viscous cream is lower. This will slow down system repsonse.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison << F2620 Comparison] | Conclusions | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T21:11:49Z<p>LucasCY: /* Infector Detector: Conclusions */</p>
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<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
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<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
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__NOTOC__<br />
=Infector Detector: Conclusion =<br />
The major achievements of the infector detecter project:<br />
*Extensive modelling of the two potential constructs for infector detector<br />
*Pufurification of GFPmut3b to allow construction of a calibration curve <br />
*Detailed characterisation of construct 1 ''in vitro'' using a calibration curve to into rate of GFP synthesis<br />
*Characterisation of the construct 1 ''in vitro'' <br />
*To create a standard unit to allow comparison between ''in vitro'' and ''in vivo''<br />
<br />
The table below summarises our infector detector system in the context of the orginial specifications that we set out to achieve:<br />
<br />
{|border="1" width="80%" align="center"<br />
|-<br />
|width="20%" style="background:#ffffcc"|<center>'''Property'''</center><br />
|width="--"|<center>'''Value'''</center><br />
|'''Achievements'''<br />
|-<br />
|style="background:#ffffcc"|Inputs<br />
|<center>System must be sensitive to AHL concentration between 5-50nM</center><br />
|'''<font color=green>Sensitive to 5-1000nM</font>'''<br />
|-<br />
|style="background:#ffffcc"|Outputs<br />
|<center>System must give a visual signal if bacteria is present</center><br />
|'''<font color=red>Future work - Using Stronger fluorescent protein such as DsRed express</font>''' <br />
|-<br />
|style="background:#ffffcc"|Response Time<br />
|<center>System needs to have a response time under 3 hour</center><br />
|'''<font color=green>Systems responds <30minutes</font>'''<br />
|-<br />
|style="background:#ffffcc"|Operating Conditions<br />
|<center>System must operate within temperature 20-30&deg;C</center><br />
|'''<font color=green>System works at 25&deg;C</font>'''<br />
|-<br />
|style="background:#ffffcc"|Health & Safety<br />
|<center>System Must not be living replicating bacteria, and in any way harmful or infectious.</center><br />
|'''<font color=green>Cell Free ''in vitro'' chassis</font>'''<br />
|-<br />
|style="background:#ffffcc"|Lifespan<br />
|<center>System must have a shelf life of 7 days</center><br />
|'''<font color=green>Can be stored in freezer for prolonged periods</font>'''<br />
|-<br />
|style="background:#ffffcc"|Packaging<br />
|<center>System must be portable and convenient to use</center><br />
|'''<font color=red>Future Work - Using our chassis in a spray</font>''' <br />
|}<br />
<br clear="all"><br />
<br />
<br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector detector is that it is not limited to just one infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. For example by using a construct that recognises AI-2<sup>[[#References |1]]</sup> , we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:IC2007 Conculsion4.jpg|thumb|right|390px|Tweaking sensitivity using LuxR]] <br />
The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br><br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity. Briefly, if we want to detect only highly progressed infections, we add little LuxR. If we want to detect infections with minimum progression, we saturate with AHL.<br><br><br />
Thus construct 2 can be used as a sensitivity attenuator.<br />
<br />
<br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged into either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as he can simply spray from a distance. The disadvantage being poor accuracy of application and more evaporation.<br />
<br />
<br />
A cream on the other hand will decrease significanlty any evaporation and will allow the user to apply the detector to specific areas of the catheter without the possibility of spraying the patient itself with detector. The disadvantage being here is the low diffusion rates of AHL and other compounds that need to cross the viscous cream to reach the actual detector assemblies. This might hinder rapid detection.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison << F2620 Comparison] | Conclusions | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Template:IC07navmenuTemplate:IC07navmenu2007-10-26T21:10:45Z<p>LucasCY: </p>
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--></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/SpecificationImperial/Infector Detector/Specification2007-10-26T20:56:23Z<p>LucasCY: /* Infector Detector: Specifications */</p>
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<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
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<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
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__NOTOC__<br />
= Infector Detector: Specifications =<br />
<br />
The system must be able to detect the presence of biofilms on urinary catheters by detection of AHL, at a minimum concentration of 5nM, and report with a visual signal within 3 hours. It must work within a temperature range of 20&deg;-30&deg;C, be portable and easy to use, have a shelf life of at least seven days, and must not be harmful or infectious.<br />
<br />
<!--<br />
<br />
{|border="1" width="80%" align="center"<br />
|-<br />
|width="20%" style="background:#ffffcc"|<center>'''Property'''</center><br />
|width="--"|<center>'''Value'''</center><br />
|-<br />
|style="background:#ffffcc"|Inputs<br />
|<center>System must be sensitive to AHL concentration between 5-50nM</center><br />
|-<br />
|style="background:#ffffcc"|Outputs<br />
|<center>System must give a visual signal if bacteria is present</center><br />
|-<br />
|style="background:#ffffcc"|Response Time<br />
|<center>System needs to have a response time under 3 hour</center><br />
|-<br />
|style="background:#ffffcc"|Operating Conditions<br />
|<center>System must operate within temperature 20-30&deg;C</center><br />
|-<br />
|style="background:#ffffcc"|Health & Safety<br />
|<center>System Must not be living replicating bacteria, and in any way harmful or infectious.</center><br />
|-<br />
|style="background:#ffffcc"|Lifespan<br />
|<center>System must have a shelf life of 7 days</center><br />
|-<br />
|style="background:#ffffcc"|Packaging<br />
|<center>System must be portable and convenient to use</center><br />
|}<br />
<br clear="all"><br />
<br />
== Specifications in detail ==<br />
<br />
--><br />
<br />
[[Image:IC07 spec sysresponse.png|thumb|400px|left|'''Desired response''' of Infector Detector. Note that the response is not necessarily linear.]]<br />
* '''<span style="color:#9933FF ; font-size:120%;">Input:</span> AHL 5-50nM'''<br />
It is known that, in Pseudomonas ''aeruginosa'' biofilms, the concentration of AHL is at least 5nM. <sup>[[#References|1-2]]</sup> Therefore, if the system can successfully report the presence of this concentration, it should be able to detect such biofilms.<br />
* '''<span style="color:#9933FF ; font-size:120%;">Output:</span> Visible fluorescent protein'''<br />
In order for the system to be used by a nurse, without needing any special equipment, the output signal must be visible.<br />
* '''<span style="color:#9933FF ; font-size:120%;">Response Time:</span> < 3 hours'''<br />
Given that biofilms grow and spread in a period of hours and days, the response time of the system must be as short as possible. The threshold of three hours is achievable in the time constraints of protein expression systems, yet it is short enough in comparison to the growth properties of biofilms. <sup>[[#References|3-6]]</sup><br />
* '''<span style="color:#9933FF ; font-size:120%;">Operating conditions:</span> Temperatures from 20&deg; to 30&deg;C'''<br />
The system will be applied to urinary catheters ''in-situ'', and therefore should function at the ambient temperature of hospitals - 20&deg; to 30&deg;C.<br />
<br clear="all"><br />
* '''<span style="color:#9933FF ; font-size:120%;">Health & safety:</span> Non-living, non-infectious'''<br />
Because the system will be applied to urinary catheters ''in-situ'', it is likely that contact with the skin of the patient will occur. It is essential that it does not cause any harm or infection to the patient. Furthermore, any living organism is susceptible to mutations that may affect its intended function, or may proliferate in the environment of the catheter and urethra.<br />
* '''<span style="color:#9933FF ; font-size:120%;">Packaging and shelf-life:</span> 7 days'''<br />
The system should be easily applied by a nurse caring for a patient with a urinary catheter. Hence, it should be packaged as a cream or a spray. In order to extend its usefulness and convenience, the system should also have a shelf-life of at least 7 days in a common hospital storage facility (shelves, cupboards, refrigerators, or freezers).<br />
<br />
<br clear="all"><br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Introduction << Introduction] | Specifications | [https://2007.igem.org/Imperial/Infector_Detector/Design Design >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Charlton, TS, et al. A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography–mass spectrometry: application to a model bacterial biofilm. Environmental Microbiology 2 (5), 530–541. October 2000.<br />
# Stickler DJ, Morris NS, McLean RJ, and Fuqua C. Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro. Appl Environ Microbiol 1998 Sep; 64(9) 3486-90.<br />
# Morris NS, Stickler DJ, and McLean RJ. The development of bacterial biofilms on indwelling urethral catheters. World J Urol 1999 Dec; 17(6) 345-50.<br />
# Drinka PJ. Complications of chronic indwelling urinary catheters. J Am Med Dir Assoc 2006 Jul; 7(6) 388-92. doi:10.1016/j.jamda.2006.01.020 pmid:16843240.<br />
# Stickler, DJ, et al. A Sensor To Detect the Early Stages in the Development of Crystalline Proteus mirabilis Biofilm on Indwelling Bladder Catheters. Journal of Clinical Microbiology, April 2006, p. 1540-1542, Vol. 44, No. 4.<br />
# [http://inls.ucsd.edu/~volfson/biofilm/ Growth and ordering of biofilms in controlled environments] available online on 22.10.2007</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:16:25Z<p>LucasCY: /* AHL Testing */</p>
<hr />
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<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:1px solid #000077; border-right:1px solid #000077; border-bottom:1px solid #000077; border-left:1px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br clear=all><br><br />
<br />
===AHL Testing===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:1px solid #000077; border-right:1px solid #000077; border-bottom:1px solid #000077; border-left:1px solid #000077;" <br />
|<br><center>[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|420px|left|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
[[Image:Titrations curve - molecules.PNG|thumb|440px|right|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]</center><br />
|-<br />
|style="text-align: left;" |<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR. <br><br><br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br><br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
|}<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:14:32Z<p>LucasCY: /* DNA Concentrations */</p>
<hr />
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<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:1px solid #000077; border-right:1px solid #000077; border-bottom:1px solid #000077; border-left:1px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br clear=all><br><br />
<br />
===AHL Testing===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|420px|left|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
[[Image:Titrations curve - molecules.PNG|thumb|440px|right|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]</center><br />
|-<br />
|style="text-align: left;" |<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR. <br><br><br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br><br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
|}<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:13:12Z<p>LucasCY: /* AHL Testing */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br clear=all><br><br />
<br />
===AHL Testing===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|420px|left|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
[[Image:Titrations curve - molecules.PNG|thumb|440px|right|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]</center><br />
|-<br />
|style="text-align: left;" |<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR. <br><br><br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br><br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
|}<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:12:04Z<p>LucasCY: /* AHL Testing */</p>
<hr />
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<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br clear=all><br><br />
<br />
===AHL Testing===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|420px|left|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
[[Image:Titrations curve - molecules.PNG|thumb|420px|right|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]</center><br />
|-<br />
|style="text-align: left;" |<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR. <br><br><br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br><br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
|}<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:11:18Z<p>LucasCY: /* AHL Testing */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br clear=all><br><br />
<br />
===AHL Testing===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|330px|left|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
[[Image:Titrations curve - molecules.PNG|thumb|440px|right|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]</center><br />
|-<br />
|<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR. <br><br><br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br><br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
|}<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:09:18Z<p>LucasCY: /* DNA Concentrations */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br clear=all><br><br />
<br />
===AHL Testing===<br />
<br />
{|align="center"<br />
| width="50%"|[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|330px|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
| width="50%"| [[Image:Titrations curve - molecules.PNG|thumb|440px|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]<br />
|}<br />
<br />
<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR.<br />
<br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br />
<br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:08:52Z<p>LucasCY: /* DNA Concentrations */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br />
===AHL Testing===<br />
<br />
{|align="center"<br />
| width="50%"|[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|330px|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
| width="50%"| [[Image:Titrations curve - molecules.PNG|thumb|440px|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]<br />
|}<br />
<br />
<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR.<br />
<br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br />
<br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/TestingImperial/Infector Detector/Testing2007-10-26T20:08:36Z<p>LucasCY: /* DNA Concentrations */</p>
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<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
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__NOTOC__<br />
= Infector Detector: Testing =<br />
==Summary==<br />
The key results of the testing were:<br />
*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.<br />
<br />
== Aims==<br />
The aims of the testing were as follows:<br />
*To test and obtain the '''optimal DNA concentration for construct 1 ''in vitro''<br />
*To characterise the '''output of GFPmut3b for a range of AHL inputs'''. From this obtain the AHL sensitivity of our system.<br />
Both of these are important, first to try to optimise infector detector to reach the full potential of ''in vitro'' chassis and secondly to the specifications for the sensitivity to AHL.<br />
<br />
In addition the fluorescence measurements were converted to number of GFPmut3b molecules synthesised using a calibration curve constructed using purified GFPmut3b.<br />
<br />
==Results==<br />
===DNA Concentrations===<br />
===DNA Concentrations===<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' <br />
[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]<br />
[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center><br />
|-<br />
|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:<br />
*'''Linear phase''' - The DNA Concentration is proportional to synthesis of GFP molecules<br />
*'''Saturation phase''' - The expressional machinary is saturated i.e. RNA polymerases and ribosomes, and so the synthesis is no longer affected by DNA concentration. The maximum synthesis of GFP is at 4&micro;g.<br />
*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.<br />
The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br><br />
For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br><br />
The 4&micro;g was taken as the maximum and used for the rest of the testing.<br />
|}<br />
<br />
===AHL Testing===<br />
<br />
{|align="center"<br />
| width="50%"|[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|330px|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]<br />
| width="50%"| [[Image:Titrations curve - molecules.PNG|thumb|440px|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]<br />
|}<br />
<br />
<br><br />
Figure 1.3 shows us the following:<br />
*The output of '''GFPmut3b increases with input of AHL'''<br />
*The system is sensitive to a range of '''5-1000nM AHL'''<br />
*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced, showing that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR.<br />
<br />
Figure 1.4 shows us the following:<br />
*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.<br />
*The '''lower threshold of response''' is the AHL concentration that the construct will respond<br />
*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br />
<br />
For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Implementation<< Implementation] | Testing | [https://2007.igem.org/Imperial/Infector_Detector/F2620_Comparison F2620 Comparison >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/F2620_ComparisonImperial/Infector Detector/F2620 Comparison2007-10-26T19:55:11Z<p>LucasCY: </p>
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<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
<br />
=Summary of Comparison=<br />
We compared our ''in vitro'' characterisation to the characterisation of [http://partsregistry.org/Part:BBa_F2620 F2620] ''in vivo'' with the aim to highlight some of the differences between the chassis and investigate how the constructs characteristics may change between them. The [http://partsregistry.org/Part:BBa_F2620 F2620] is an ideal construct to compare for comparison because of its detailed characterisation ''in vivo''. The construct is the same as the construct 1 that was used for infecter detector, '''pTet-LuxR-pLux-GFPmut3b'''. The key results the comparison were;<br />
*The creation of a new unit to allow comparison between ''in vitro'' and ''in vivo'' chassis.<br />
*The constructs response appears to be largely independent of the chassis used.<br />
Both of these are important findings and highlight the potential that new chassis offer to synthetic biology.<br />
<br />
<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<center>[[image:IC2007 BF stage1 construct.PNG |700px]]</center><br />
{|style="width:800px; text-align: left;" border="0" <br />
|<br>The basis for comparison is to normalise the ''in vitro'' chassis on the number of plasmids to give a platform for comparison:<br />
*''In Vitro'' - 4&micro;g of DNA was added which for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] is 904823007 plasmids<br />
*''In Vivo'' - Each cell the plasmids number was estimated at 30 per cell<br />
To compare we normalised the data of ''in vitro'' '''GFPmut3b molecules synthesised per 30 plasmids''' to allow some comparison to the ''in vivo'' data. <br />
<br />
Of particular interest was to compare the:<br />
#'''Rate of GFP synthesis''' of 100nM<br />
#'''Transfer Function'''.<br />
|}<br />
|}<br />
<br />
<br clear=all><br />
{|align="center" style="width:800px; text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;"<br />
|<center>[[image:In vitro in vivo comp.png|700px]]</center><br />
|-<br />
|style="text-align: left;" |Comparison between ''in vivo'' and ''in vitro'' for rate of GFPmut3b synthesis for 100nM AHL. The ''in vivo'' chassis used was the bacterial strain MG1655 and the ''in vitro'' chassis was Promega Commercial S30 Cell Extract.<br />
<br><br />
The graph shows the following:<br />
*''in vivo'' has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. In addition the rate reaches a steady state after around 30minutes and maintains it for the duration of the testing.<br><br />
*''in vitro'' has the equivalent of 220 molecules of GFP synthesised per second per cell equivalent, the cell equilavent being based upon the normalization of DNA plasmids. Interestingly the ''in vitro'' chassis does not reach a steady state, in fact it decreases in rate of synthesis after 90 minutes and keeps decreasing until rate is zero at around 360 minutes.<br />
<br><br />
The reason why the ''in vitro'' chassis never reaches a steady state is because of the limited energy and metabolites available, this is unlike ''in vivo'' which is supported by the media that it is grown upon.<br />
<br><br />
Interestingly the values of rate of synthesis are in the same order magnitude of hundreds, this suggesting that the normalisation we are using to compare these chassis is valid.<br />
|}<br />
<br />
===Transfer Function===<br />
<br />
<br clear=all><br />
{|align="center" style="width:800px; text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<center>[[image:In_vivo_in_vitro_comp2.png|700px]]</center><br />
|-<br />
|style="text-align: left;" |The graph above shows the transfer function of '''[AHL] <font color=red>input</font>''' vs '''rate of GFP synthesis <font color=red>output</font>'''. The graph shows the max rate of synthesis for each of the chassis; for ''in vivo'' this is the steady state reached after about 30 minutes and for ''in vitro'' it is the rate between 60 and 90 minutes which is the maximum rate before the energy limitations of the system cause the rate to drop. The blue line on corresponds to the range of AHL and the response of the ''in vitro'' chassis.<br><br />
<br><br />
The key difference between the chassis is the rate of GFP synthesis which is lower in the ''in vitro'' chassis e.g. for 1000nM of AHL the rate of GFP synthesis ''in vivo'' is ~450 GFP molecules per sec per cell,'' ''in vitro'' has an equivalent value of 220 GFP molecules per second.<br><br />
<br><br />
The shape of the transfer function is very similar for both chassis, both begin to saturate at around 1000nM of AHL and the threshold of sensitivity is around 1nM AHL. It is very surprising that a construct works so similar in different chassis, showing the affect of the chassis is minimal to the constructs behavior. <br />
|}<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Testing<< Testing] | F2620 Comparison| [https://2007.igem.org/Imperial/Infector_Detector/Conclusion Conclusions >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/F2620_ComparisonImperial/Infector Detector/F2620 Comparison2007-10-26T19:48:57Z<p>LucasCY: </p>
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<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
<br />
=Summary of Comparison=<br />
We compared our ''in vitro'' characterisation to the characterisation of [http://partsregistry.org/Part:BBa_F2620 F2620] ''in vivo'' with the aim to highlight some of the differences between the chassis and investigate how the constructs characteristics may change between them. The [http://partsregistry.org/Part:BBa_F2620 F2620] is an ideal construct to compare for comparison because of its detailed characterisation ''in vivo''. The construct is the same as the construct 1 that was used for infecter detector, '''pTet-LuxR-pLux-GFPmut3b'''. The key results the comparison were;<br />
*The creation of a new unit to allow comparison between ''in vitro'' and ''in vivo'' chassis.<br />
*The constructs response appears to be largely independent of the chassis used.<br />
Both of these are important findings and highlight the potential that new chassis offer to synthetic biology.<br />
<br />
<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<center>[[image:IC2007 BF stage1 construct.PNG |700px]]</center><br />
{|style="width:800px; text-align: left;" border="0" <br />
|<br>The basis for comparison is to normalise the ''in vitro'' chassis on the number of plasmids to give a platform for comparison:<br />
*''In Vitro'' - 4&micro;g of DNA was added which for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] is 904823007 plasmids<br />
*''In Vivo'' - Each cell the plasmids number was estimated at 30 per cell<br />
To compare we normalised the data of ''in vitro'' '''GFPmut3b molecules synthesised per 30 plasmids''' to allow some comparison to the ''in vivo'' data. <br />
<br />
Of particular interest was to compare the:<br />
#'''Rate of GFP synthesis''' of 100nM<br />
#'''Transfer Function'''.<br />
|}<br />
|}<br />
<br />
<br clear=all><br />
{|align="center" style="width:800px; text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;"<br />
|<center>[[image:In vitro in vivo comp.png|700px]]</center><br />
|-<br />
|style="text-align: left;" |Comparison between ''in vivo'' and ''in vitro'' for rate of GFPmut3b synthesis for 100nM AHL. The ''in vivo'' chassis used was the bacterial strain MG1655 and the ''in vitro'' chassis was Promega Commercial S30 Cell Extract.<br />
<br><br />
The graph shows the following:<br />
*''in vivo'' has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. In addition the rate reaches a steady state after around 30minutes and maintains it for the duration of the testing.<br><br />
*''in vitro'' has the equivalent of 220 molecules of GFP synthesised per second per cell equivalent, the cell equilavent being based upon the normalization of DNA plasmids. Interestingly the ''in vitro'' chassis does not reach a steady state, in fact it decreases in rate of synthesis after 90 minutes and keeps decreasing until rate is zero at around 360 minutes.<br />
<br><br />
The reason why the ''in vitro'' chassis never reaches a steady state is because of the limited energy and metabolites available, this is unlike ''in vivo'' which is supported by the media that it is grown upon.<br />
<br><br />
Interestingly the values of rate of synthesis are in the same order magnitude of hundreds, this suggesting that the normalisation we are using to compare these chassis is valid.<br />
|}<br />
<br />
===Transfer Function===<br />
{|align="center"<br />
|width="100%"|<br>[[image:In_vivo_in_vitro_comp2.png|thumb|800px|The graph above shows the transfer function of '''[AHL] <font color=red>input</font>''' vs '''rate of GFP synthesis <font color=red>output</font>'''. The graph shows the max rate of synthesis for each of the chassis; for ''in vivo'' this is the steady state reached after about 30 minutes and for ''in vitro'' it is the rate between 60 and 90 minutes which is the maximum rate before the energy limitations of the system cause the rate to drop. The blue line on corresponds to the range of AHL and the response of the ''in vitro'' chassis.<br><br />
<br><br />
The key difference between the chassis is the rate of GFP synthesis which is lower in the ''in vitro'' chassis e.g. for 1000nM of AHL the rate of GFP synthesis ''in vivo'' is ~450 GFP molecules per sec per cell,'' ''in vitro'' has an equivalent value of 220 GFP molecules per second.<br><br />
<br><br />
The shape of the transfer function is very similar for both chassis, both begin to saturate at around 1000nM of AHL and the threshold of sensitivity is around 1nM AHL. It is very surprising that a construct works so similar in different chassis, showing the affect of the chassis is minimal to the constructs behavior. <br />
]]<br />
|}<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Testing<< Testing] | F2620 Comparison| [https://2007.igem.org/Imperial/Infector_Detector/Conclusion Conclusions >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/F2620_ComparisonImperial/Infector Detector/F2620 Comparison2007-10-26T19:39:57Z<p>LucasCY: </p>
<hr />
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<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/F2620 Comparison" title=""><span>F2620</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
<br />
=Summary of Comparison=<br />
We compared our ''in vitro'' characterisation to the characterisation of [http://partsregistry.org/Part:BBa_F2620 F2620] ''in vivo'' with the aim to highlight some of the differences between the chassis and investigate how the constructs characteristics may change between them. The [http://partsregistry.org/Part:BBa_F2620 F2620] is an ideal construct to compare for comparison because of its detailed characterisation ''in vivo''. The construct is the same as the construct 1 that was used for infecter detector, '''pTet-LuxR-pLux-GFPmut3b'''. The key results the comparison were;<br />
*The creation of a new unit to allow comparison between ''in vitro'' and ''in vivo'' chassis.<br />
*The constructs response appears to be largely independent of the chassis used.<br />
Both of these are important findings and highlight the potential that new chassis offer to synthetic biology.<br />
<br />
<br />
<br clear=all><br />
{|align="center" style="text-align: center; border-top:2px solid #000077; border-right:2px solid #000077; border-bottom:2px solid #000077; border-left:2px solid #000077;" <br />
|<center>[[image:IC2007 BF stage1 construct.PNG |700px]]</center><br />
{|style="width:800px; text-align: left;" border="0" <br />
|<br>The basis for comparison is to normalise the ''in vitro'' chassis on the number of plasmids to give a platform for comparison:<br />
*''In Vitro'' - 4&micro;g of DNA was added which for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] is 904823007 plasmids<br />
*''In Vivo'' - Each cell the plasmids number was estimated at 30 per cell<br />
To compare we normalised the data of ''in vitro'' '''GFPmut3b molecules synthesised per 30 plasmids''' to allow some comparison to the ''in vivo'' data. <br />
<br />
Of particular interest was to compare the:<br />
#'''Rate of GFP synthesis''' of 100nM<br />
#'''Transfer Function'''.<br />
|}<br />
|}<br />
<br />
<br><br />
{|align="center"<br />
|width="100%"|<br>[[image:In vitro in vivo comp.png|thumb|800px|Comparison between ''in vivo'' and ''in vitro'' for rate of GFPmut3b synthesis for 100nM AHL. The ''in vivo'' chassis used was the bacterial strain MG1655 and the ''in vitro'' chassis was Promega Commercial S30 Cell Extract.<br />
<br><br />
The graph shows the following:<br />
*''in vivo'' has a maximal rate of 400-500 molecules of GFP synthesised per second per cell. In addition the rate reaches a steady state after around 30minutes and maintains it for the duration of the testing.<br><br />
*''in vitro'' has the equivalent of 220 molecules of GFP synthesised per second per cell equivalent, the cell equilavent being based upon the normalization of DNA plasmids. Interestingly the ''in vitro'' chassis does not reach a steady state, in fact it decreases in rate of synthesis after 90 minutes and keeps decreasing until rate is zero at around 360 minutes.<br />
<br><br />
The reason why the ''in vitro'' chassis never reaches a steady state is because of the limited energy and metabolites available, this is unlike ''in vivo'' which is supported by the media that it is grown upon.<br />
<br><br />
Interestingly the values of rate of synthesis are in the same order magnitude of hundreds, this suggesting that the normalisation we are using to compare these chassis is valid.<br />
]]<br />
|}<br />
<br />
===Transfer Function===<br />
{|align="center"<br />
|width="100%"|<br>[[image:In_vivo_in_vitro_comp2.png|thumb|800px|The graph above shows the transfer function of '''[AHL] <font color=red>input</font>''' vs '''rate of GFP synthesis <font color=red>output</font>'''. The graph shows the max rate of synthesis for each of the chassis; for ''in vivo'' this is the steady state reached after about 30 minutes and for ''in vitro'' it is the rate between 60 and 90 minutes which is the maximum rate before the energy limitations of the system cause the rate to drop. The blue line on corresponds to the range of AHL and the response of the ''in vitro'' chassis.<br><br />
<br><br />
The key difference between the chassis is the rate of GFP synthesis which is lower in the ''in vitro'' chassis e.g. for 1000nM of AHL the rate of GFP synthesis ''in vivo'' is ~450 GFP molecules per sec per cell,'' ''in vitro'' has an equivalent value of 220 GFP molecules per second.<br><br />
<br><br />
The shape of the transfer function is very similar for both chassis, both begin to saturate at around 1000nM of AHL and the threshold of sensitivity is around 1nM AHL. It is very surprising that a construct works so similar in different chassis, showing the affect of the chassis is minimal to the constructs behavior. <br />
]]<br />
|}<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Testing<< Testing] | F2620 Comparison| [https://2007.igem.org/Imperial/Infector_Detector/Conclusion Conclusions >>]<br />
</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-09-14Imperial/Wet Lab/Lab Notebook/2007-09-142007-10-26T19:01:36Z<p>LucasCY: /* Agarose Gel Electrophoresis of Biobricks */</p>
<hr />
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__NOTOC__<br />
= 14 September 2007 =<br />
<br />
<br />
==Restriction Digest of Biobricks==<br />
#4 DNA samples were digested by SpeI and PstI<br />
#* Samples 5, 7, 24 <br />
#1 DNA sample wasdigested by XbaI and PstI<br />
#* Samples 23<br />
#4 DNA samples were digested by EcoRI<br />
#* Samples 13, 25, 27b-e, T7, Kirsten's parts<br />
#4 μl of DNA was digested by 1 μl of enzymes at 37&deg;C for 1 hour<br>Exception: Sample 23 had 6 μl DNA and 2 μl enzymes<br />
<br />
Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#Restriction_Digest | Restriction Digest] in the general protocols page<br />
<br />
==Agarose Gel Electrophoresis of Biobricks==<br />
#Checked 9 Biobricks and 13 Digests on 1% agarose gel <br />
[[Image:ICGEMS Gel 14 9.png|900px|]]<br />
<br />
Conclusions:<br />
*Part 27 is confirmed to be faulty<br />
*T7 maxi-prep was not done properly<br />
*Kirsten's parts were most likely not there...<br />
<br />
Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#Electrophoresis | Electrophoresis] in the general protocols page<br />
<br />
==Gel Purification of Biobricks==<br />
<br />
#Gel was melted and run through column<br />
#Purified DNA was eluted with ddH<sub>2</sub>O<br />
<font size=-2><br />
*5. BBa_R0040 [ptet] (~2100 bp fragment)<br />
*7. BBa_F2620 [ptet-LuxR-pLux] (~3100 bp fragment)<br />
*23. BBa_I13504 [GFP] (~800 bp fragment)<br />
*24. BBa_R0062 [pLux] (~2100 bp fragment)<br />
</font><br />
<br />
Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#Gel_Purification | Gel Purification] in the general protocols page<br />
<br />
<br />
<html></div></html><br />
{{Template:IC07labnotebook}}</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-19Imperial/Wet Lab/Lab Notebook/2007-10-192007-10-26T18:59:58Z<p>LucasCY: </p>
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<div>{{Template:IC07navmenu}}<br />
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__NOTOC__<br />
<br />
<br />
==AHL titration curve for infector detector construct==<br />
*The '''aim'' of this experiment was to test the pTet-luxR-pLux-GFPmut3b construct with a range of AHL concentrations. A titration curve could then be created for the construct in terms of AHL concentrations<br />
*The materials and methods can be found in the [[Imperial/Wet Lab/Protocols/ID3.1|Protocols]]<br />
*The concentrations of AHL for which the construct was tested are: 0nM, 5nM, 10nM, 15nM, 20nM, 50nM, 100nM, 1000nM<br />
*There were two negative controls for the experiment:<br />
**Empty vector with [AHL]=1000nM<br />
**pTet-luxR-pLux-GFPmut3b construct with [AHL] = 0nM<br />
*The [[Imperial/Wet Lab/Results/ID3.1|Results]] showed that the total fluorescent output produced by the construct increases with increasing [AHL]<br />
<br />
<br />
<br />
<html></div></html><br />
{{Template:IC07labnotebook}}</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-19Imperial/Wet Lab/Lab Notebook/2007-10-192007-10-26T18:58:32Z<p>LucasCY: </p>
<hr />
<div><html></div></html><br />
{{Template:IC07labnotebook}}<br />
<br />
<br />
==AHL titration curve for infector detector construct==<br />
*The '''aim'' of this experiment was to test the pTet-luxR-pLux-GFPmut3b construct with a range of AHL concentrations. A titration curve could then be created for the construct in terms of AHL concentrations<br />
*The materials and methods can be found in the [[Imperial/Wet Lab/Protocols/ID3.1|Protocols]]<br />
*The concentrations of AHL for which the construct was tested are: 0nM, 5nM, 10nM, 15nM, 20nM, 50nM, 100nM, 1000nM<br />
*There were two negative controls for the experiment:<br />
**Empty vector with [AHL]=1000nM<br />
**pTet-luxR-pLux-GFPmut3b construct with [AHL] = 0nM<br />
*The [[Imperial/Wet Lab/Results/ID3.1|Results]] showed that the total fluorescent output produced by the construct increases with increasing [AHL]<br />
<br />
<br />
<br />
<html></div></html><br />
{{Template:IC07labnotebook}}</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-19Imperial/Wet Lab/Lab Notebook/2007-10-192007-10-26T18:58:18Z<p>LucasCY: </p>
<hr />
<div><html></div></html><br />
{{Template:IC07labnotebook}}<br />
<br />
==AHL titration curve for infector detector construct==<br />
*The '''aim'' of this experiment was to test the pTet-luxR-pLux-GFPmut3b construct with a range of AHL concentrations. A titration curve could then be created for the construct in terms of AHL concentrations<br />
*The materials and methods can be found in the [[Imperial/Wet Lab/Protocols/ID3.1|Protocols]]<br />
*The concentrations of AHL for which the construct was tested are: 0nM, 5nM, 10nM, 15nM, 20nM, 50nM, 100nM, 1000nM<br />
*There were two negative controls for the experiment:<br />
**Empty vector with [AHL]=1000nM<br />
**pTet-luxR-pLux-GFPmut3b construct with [AHL] = 0nM<br />
*The [[Imperial/Wet Lab/Results/ID3.1|Results]] showed that the total fluorescent output produced by the construct increases with increasing [AHL]<br />
<br />
<br />
<html></div></html><br />
{{Template:IC07labnotebook}}</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-19Imperial/Wet Lab/Lab Notebook/2007-10-192007-10-26T18:57:59Z<p>LucasCY: </p>
<hr />
<div><html></div></html><br />
{{Template:IC07labnotebook}}<br />
<br />
==AHL titration curve for infector detector construct==<br />
*The '''aim'' of this experiment was to test the pTet-luxR-pLux-GFPmut3b construct with a range of AHL concentrations. A titration curve could then be created for the construct in terms of AHL concentrations<br />
*The materials and methods can be found in the [[Imperial/Wet Lab/Protocols/ID3.1|Protocols]]<br />
*The concentrations of AHL for which the construct was tested are: 0nM, 5nM, 10nM, 15nM, 20nM, 50nM, 100nM, 1000nM<br />
*There were two negative controls for the experiment:<br />
**Empty vector with [AHL]=1000nM<br />
**pTet-luxR-pLux-GFPmut3b construct with [AHL] = 0nM<br />
*The [[Imperial/Wet Lab/Results/ID3.1|Results]] showed that the total fluorescent output produced by the construct increases with increasing [AHL]</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-17Imperial/Wet Lab/Lab Notebook/2007-10-172007-10-26T18:57:46Z<p>LucasCY: </p>
<hr />
<div>{{Template:IC07navmenu}}<br />
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__NOTOC__<br />
<br />
<br />
==Temperature step test for pTet-GFPmut3b==<br />
*The '''aim''' of this test was to determine the response of the pTet-GFPmut3b construct to a step change in temperature.<br />
*For complete materials and methods see the [[Imperial/Wet_Lab/Protocols/CBD2.3|Protocol]] <br />
*The construct was tested at two temperature steps:<br />
*#From 4&deg;C (for 3 hours) to 20&deg;C (3 hours)<br />
*#From 20&deg;C (for 3 hours) to 4&deg;C (3 hours)<br />
*The sampling time was every hour<br />
*The [[Imperial/Wet_Lab/Results/CBD2.3|Results]] show that the step changes in temperature affect the fluorescence produced by the pTet-GFPmut3b construct<br />
<br />
<br />
<html></div></html><br />
{{Template:IC07labnotebook}}</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-17Imperial/Wet Lab/Lab Notebook/2007-10-172007-10-26T18:57:13Z<p>LucasCY: </p>
<hr />
<div><html></div></html><br />
{{Template:IC07labnotebook}}<br />
<br />
{{Template:IC07navmenu}}<br />
<html><div id="maincol"></html><br />
__NOTOC__<br />
<br />
<br />
==Temperature step test for pTet-GFPmut3b==<br />
*The '''aim''' of this test was to determine the response of the pTet-GFPmut3b construct to a step change in temperature.<br />
*For complete materials and methods see the [[Imperial/Wet_Lab/Protocols/CBD2.3|Protocol]] <br />
*The construct was tested at two temperature steps:<br />
*#From 4&deg;C (for 3 hours) to 20&deg;C (3 hours)<br />
*#From 20&deg;C (for 3 hours) to 4&deg;C (3 hours)<br />
*The sampling time was every hour<br />
*The [[Imperial/Wet_Lab/Results/CBD2.3|Results]] show that the step changes in temperature affect the fluorescence produced by the pTet-GFPmut3b construct<br />
<br />
<html></div></html><br />
{{Template:IC07labnotebook}}</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-17Imperial/Wet Lab/Lab Notebook/2007-10-172007-10-26T18:56:02Z<p>LucasCY: </p>
<hr />
<div><html></div></html><br />
{{Template:IC07labnotebook}}<br />
<br />
==Temperature step test for pTet-GFPmut3b==<br />
*The '''aim''' of this test was to determine the response of the pTet-GFPmut3b construct to a step change in temperature.<br />
*For complete materials and methods see the [[Imperial/Wet_Lab/Protocols/CBD2.3|Protocol]] <br />
*The construct was tested at two temperature steps:<br />
*#From 4&deg;C (for 3 hours) to 20&deg;C (3 hours)<br />
*#From 20&deg;C (for 3 hours) to 4&deg;C (3 hours)<br />
*The sampling time was every hour<br />
*The [[Imperial/Wet_Lab/Results/CBD2.3|Results]] show that the step changes in temperature affect the fluorescence produced by the pTet-GFPmut3b construct</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-16Imperial/Wet Lab/Lab Notebook/2007-10-162007-10-26T18:55:46Z<p>LucasCY: </p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><div id="maincol"></html><br />
__NOTOC__<br />
<br />
<br />
==pT7-GFPmut3b chracterisation==<br />
*The '''aim''' of today's test was to chracterise the pT7-GFPmut3b with respect to temperature<br />
*For materials and methods see the [[Imperial/Wet_Lab/Protocols/Prot1.9|Protocol]]<br />
*The construct was tested for three temperatures: 4&deg;C, 25&deg;C and 37&deg;C <br />
*The reactions were sampled every hour<br />
*The [[Imperial/Wet_Lab/Results/Res1.9|Results]] showed that pT7-GFPmut3b expressed at all three temperatures and the fluorescence is the highest for greater temperatures<br />
<br />
<html></div></html><br />
{{Template:IC07labnotebook}}</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-19Imperial/Wet Lab/Lab Notebook/2007-10-192007-10-26T18:54:55Z<p>LucasCY: </p>
<hr />
<div>==AHL titration curve for infector detector construct==<br />
*The '''aim'' of this experiment was to test the pTet-luxR-pLux-GFPmut3b construct with a range of AHL concentrations. A titration curve could then be created for the construct in terms of AHL concentrations<br />
*The materials and methods can be found in the [[Imperial/Wet Lab/Protocols/ID3.1|Protocols]]<br />
*The concentrations of AHL for which the construct was tested are: 0nM, 5nM, 10nM, 15nM, 20nM, 50nM, 100nM, 1000nM<br />
*There were two negative controls for the experiment:<br />
**Empty vector with [AHL]=1000nM<br />
**pTet-luxR-pLux-GFPmut3b construct with [AHL] = 0nM<br />
*The [[Imperial/Wet Lab/Results/ID3.1|Results]] showed that the total fluorescent output produced by the construct increases with increasing [AHL]</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-17Imperial/Wet Lab/Lab Notebook/2007-10-172007-10-26T18:54:11Z<p>LucasCY: </p>
<hr />
<div>==Temperature step test for pTet-GFPmut3b==<br />
*The '''aim''' of this test was to determine the response of the pTet-GFPmut3b construct to a step change in temperature.<br />
*For complete materials and methods see the [[Imperial/Wet_Lab/Protocols/CBD2.3|Protocol]] <br />
*The construct was tested at two temperature steps:<br />
*#From 4&deg;C (for 3 hours) to 20&deg;C (3 hours)<br />
*#From 20&deg;C (for 3 hours) to 4&deg;C (3 hours)<br />
*The sampling time was every hour<br />
*The [[Imperial/Wet_Lab/Results/CBD2.3|Results]] show that the step changes in temperature affect the fluorescence produced by the pTet-GFPmut3b construct</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-10-16Imperial/Wet Lab/Lab Notebook/2007-10-162007-10-26T18:53:39Z<p>LucasCY: </p>
<hr />
<div>==pT7-GFPmut3b chracterisation==<br />
*The '''aim''' of today's test was to chracterise the pT7-GFPmut3b with respect to temperature<br />
*For materials and methods see the [[Imperial/Wet_Lab/Protocols/Prot1.9|Protocol]]<br />
*The construct was tested for three temperatures: 4&deg;C, 25&deg;C and 37&deg;C <br />
*The reactions were sampled every hour<br />
*The [[Imperial/Wet_Lab/Results/Res1.9|Results]] showed that pT7-GFPmut3b expressed at all three temperatures and the fluorescence is the highest for greater temperatures</div>LucasCYhttp://2007.igem.org/wiki/index.php/Template:IC07labnotebookTemplate:IC07labnotebook2007-10-26T18:51:32Z<p>LucasCY: </p>
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<h2><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook" title="">Lab Notebook</a></h2><br><br />
<p><br />
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<caption class="cal">July</caption><br />
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<th class="cal" scope="col" abbr="Sunday" title="Sunday">S</th><br />
<th class="cal" scope="col" abbr="Monday" title="Monday">M</th><br />
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<td class="cal">1</td><br />
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<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-07-31">31</a></td><br />
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<caption class="cal">August</caption><br />
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<th class="cal" scope="col" abbr="Sunday" title="Sunday">S</th><br />
<th class="cal" scope="col" abbr="Monday" title="Monday">M</th><br />
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<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-02">2</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-03">3</a></td><br />
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<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-06">6</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-07">7</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-08">8</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-09">9</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-10">10</a></td><br />
<td class="cal">11</td><br />
</tr><br />
<tr><br />
<td class="cal">12</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-13">13</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-14">14</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-15">15</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-16">16</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-17">17</a></td><br />
<td class="cal">18</td><br />
</tr><br />
<tr><br />
<td class="cal">19</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-20">20</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-21">21</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-22">22</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-23">23</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-24">24</a></td><br />
<td class="cal">25</td><br />
</tr><br />
<tr><br />
<td class="cal">26</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-27">27</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-28">28</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-29">29</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-30">30</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-08-31">31</a></td><br />
<td class="cal">&nbsp;</td><br />
</tr><br />
<br />
</table><br />
<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<br />
<table id="calendar" cellspacing="0" cellpadding="0"><br />
<caption class="cal">September</caption><br />
<br />
<tr><br />
<th class="cal" scope="col" abbr="Sunday" title="Sunday">S</th><br />
<th class="cal" scope="col" abbr="Monday" title="Monday">M</th><br />
<th class="cal" scope="col" abbr="Tuesday" title="Tuesday">T</th><br />
<th class="cal" scope="col" abbr="Wednesday" title="Wednesday">W</th><br />
<th class="cal" scope="col" abbr="Thursday" title="Thursday">T</th><br />
<th class="cal" scope="col" abbr="Friday" title="Friday">F</th><br />
<th class="cal" scope="col" abbr="Saturday" title="Saturday">S</th><br />
</tr><br />
<tr><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-01">1</a></td><br />
</tr><br />
<tr><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-02">2</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-03">3</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-04">4</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-05">5</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-06">6</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-07">7</a></td><br />
<td class="cal">8</td><br />
</tr><br />
<tr><br />
<td class="cal">9</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-10">10</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-11">11</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-12">12</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-13">13</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-14">14</a></td><br />
<td class="cal">15</td><br />
</tr><br />
<tr><br />
<td class="cal">16</td><br />
<td class="cal">17</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-18">18</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-19">19</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-20">20</a></td><br />
<td class="cal">21</td><br />
<td class="cal">22</td><br />
</tr><br />
<tr><br />
<td class="cal">23</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-24">24</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-25">25</a></td><br />
<td class="cal">26</td><br />
<td class="cal">27</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-09-28">28</a></td><br />
<td class="cal">29</td><br />
</tr><br />
<tr><br />
<td class="cal">30</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
</tr><br />
<br />
<br />
</table><br />
<br />
</td><br />
<td style="vertical-align: top;"><br />
<br />
<table id="calendar" cellspacing="0" cellpadding="0"><br />
<caption class="cal">October</caption><br />
<br />
<tr><br />
<th class="cal" scope="col" abbr="Sunday" title="Sunday">S</th><br />
<th class="cal" scope="col" abbr="Monday" title="Monday">M</th><br />
<th class="cal" scope="col" abbr="Tuesday" title="Tuesday">T</th><br />
<th class="cal" scope="col" abbr="Wednesday" title="Wednesday">W</th><br />
<th class="cal" scope="col" abbr="Thursday" title="Thursday">T</th><br />
<th class="cal" scope="col" abbr="Friday" title="Friday">F</th><br />
<th class="cal" scope="col" abbr="Saturday" title="Saturday">S</th><br />
</tr><br />
<tr><br />
<td class="cal">&nbsp;</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-01">1</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-02">2</a></td><br />
<td class="cal">3</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-04">4</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-05">5</a></td><br />
<td class="cal">6</td><br />
</tr><br />
<tr><br />
<td class="cal">7</td><br />
<td class="cal">8</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-09">9</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-10">10</a></td><br />
<td class="cal">11</td><br />
<td class="cal">12</td><br />
<td class="cal">13</td><br />
</tr><br />
<tr><br />
<td class="cal">14</td><br />
<td class="cal">15</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-16">16</a></td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-17">17</a></td><br />
<td class="cal">18</td><br />
<td class="cal"><a href="https://2007.igem.org/Imperial/Wet_Lab/Lab_Notebook/2007-10-19">19</a></td><br />
<td class="cal">20</td><br />
</tr><br />
<tr><br />
<td class="cal">21</td><br />
<td class="cal">22</td><br />
<td class="cal">23</td><br />
<td class="cal">24</td><br />
<td class="cal">25</td><br />
<td class="cal">26</td><br />
<td class="cal">27</td><br />
</tr><br />
<tr><br />
<td class="cal">28</td><br />
<td class="cal">29</td><br />
<td class="cal">30</td><br />
<td class="cal">31</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
<td class="cal">&nbsp;</td><br />
</tr><br />
<br />
</table><br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
</p><br />
</div><br />
</div><br />
<!-- End of Side Bar Content --><br />
<br />
<br />
</div><br />
</html></div>LucasCYhttp://2007.igem.org/wiki/index.php/User:LucasCYUser:LucasCY2007-10-26T15:44:20Z<p>LucasCY: </p>
<hr />
<div>[[Image:IC2007 IDpack1.jpg|left| A clean catheter]] [[Image:IC2007 IDpack2.jpg|right| An infected catheter]]</div>LucasCYhttp://2007.igem.org/wiki/index.php/File:IC2007_IDpack2.jpgFile:IC2007 IDpack2.jpg2007-10-26T15:40:21Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/File:IC2007_IDpack1.jpgFile:IC2007 IDpack1.jpg2007-10-26T15:39:00Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Cell_by_Date/ConclusionImperial/Cell by Date/Conclusion2007-10-26T15:08:29Z<p>LucasCY: </p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Cell_by_Date/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Cell by Date: Conclusion =<br />
<br />
Cell By Date is a great example of how synthetic biology can be integrated into our daily lives. When fully developed it can mimic the function of inducstrial level devices in the food industry at a fraction of the cost. Being a reporter of a break in the cold chain, CBD, can inform the end consumer whether the product he is buying has been exposed to abnormal storage conditions.<br />
<br />
This is best explained with an example. Imagine your local supermarket expecting a delivery of frozen lamb steaks. The delivery van arrives, the goods are unloaded and placed on the shelves awaiting for their first victim to arrive. Victim because what the supermarket manager and the end consumer are not aware of is that during their transport the van fridge broke down and thus the steaks were defrosted. The vab driver however fixed the fault, refroze the meat and told nothing upon his arrival to the supermarket. The whole incident will be kept under cover until the first cases of food poisoning arise from the comprimised lamb meat.<br />
<br />
This is were CBD comes in handy. By embedding a sticker of CBD on packaged goods, the consumer can check at any time whether the product quality has been comprimised by exposure to ubnormally high temperatures. Just a glance at the sticker and its bright fluorescent colour will inform the buyer of a potential risk. That is if the quality control expert of the supermarket does not spot it first.<br />
<br />
Even though not fully characterised, promising progress has been made and shows that even without tweaking the initial design, Cell by Date has a great potential just waiting to be exploited.<br />
<br />
= Future Work =<br />
<br />
===Extending the life of the reaction===<br />
<br />
A major constrain of CBD is its short lifetime. The cell-free extract used currently can only support it for a maximum of a few days or for the production of 12mg of protein whichever comes first. A good amount of research can go into developing cell extract that can be stored at various temperatures for months and yet have a good performance and produce a fai amount of protein. The more fluorescent protein produced the better the visible signal CBD will provide.<br />
<br />
===Packaging===<br />
<br />
Being cell-free, Cell by Date, makes it easier to be embedded on food packaging. This calls for an efficient packaging method that is transparent (for the person to be able to see the fluorescent protein) but also well isolated from the actual food source (to avoid contamination). There are also 2 important factors that need to be taken into consideration. The first is the fact that the cell extract requires oxygen for the reaction to occur. What is not known is how much is needed. This raises the issue whether sealing CBD in a package with limited oxygen will prevent the reaction from occuring. The other factor is evaporation. The smaller the sample tested, the more the evaporation there was and the quicker the reaction mixture vanished. The packaging must therefore sustain the volume of the reaction for the lifespan of the food product.<br />
<br clear="all"><br />
[[Image:IC2007 CBD packaging.jpg|thumb|left|430px|'''CBD deactivated''' - Cold chain has not been disrupted]]<br />
[[Image:IC2007 CBD packaging2.jpg|thumb|right|430px|'''CBD activated''' (Red Glow) - Cold chain has been disrupted]]<br />
<br />
[[Image:IC2007 CBD packaging3.jpg|thumb|left|430px|'''CBD deactivated''' - Cold chain has not been disrupted]]<br />
[[Image:IC2007 CBD packaging4.jpg|thumb|right|430px|'''CBD activated''' (Red Glow) - Cold chain has been disrupted]]<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Cell_by_Date/Testing << Testing ] | Conclusions | [https://2007.igem.org/Imperial Home >> ]</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/File:IC2007_CBD_packaging4.jpgFile:IC2007 CBD packaging4.jpg2007-10-26T15:07:15Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/File:IC2007_CBD_packaging3.jpgFile:IC2007 CBD packaging3.jpg2007-10-26T15:06:23Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Cell_by_Date/ConclusionImperial/Cell by Date/Conclusion2007-10-26T14:41:39Z<p>LucasCY: /* Packaging */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Cell_by_Date/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Cell by Date: Conclusion =<br />
<br />
Cell By Date is a great example of how synthetic biology can be integrated into our daily lives. When fully developed it can mimic the function of inducstrial level devices in the food industry at a fraction of the cost. Being a reporter of a break in the cold chain, CBD, can inform the end consumer whether the product he is buying has been exposed to abnormal storage conditions.<br />
<br />
This is best explained with an example. Imagine your local supermarket expecting a delivery of frozen lamb steaks. The delivery van arrives, the goods are unloaded and placed on the shelves awaiting for their first victim to arrive. Victim because what the supermarket manager and the end consumer are not aware of is that during their transport the van fridge broke down and thus the steaks were defrosted. The vab driver however fixed the fault, refroze the meat and told nothing upon his arrival to the supermarket. The whole incident will be kept under cover until the first cases of food poisoning arise from the comprimised lamb meat.<br />
<br />
This is were CBD comes in handy. By embedding a sticker of CBD on packaged goods, the consumer can check at any time whether the product quality has been comprimised by exposure to ubnormally high temperatures. Just a glance at the sticker and its bright fluorescent colour will inform the buyer of a potential risk. That is if the quality control expert of the supermarket does not spot it first.<br />
<br />
Even though not fully characterised, promising progress has been made and shows that even without tweaking the initial design, Cell by Date has a great potential just waiting to be exploited.<br />
<br />
= Future Work =<br />
<br />
===Extending the life of the reaction===<br />
<br />
A major constrain of CBD is its short lifetime. The cell-free extract used currently can only support it for a maximum of a few days or for the production of 12mg of protein whichever comes first. A good amount of research can go into developing cell extract that can be stored at various temperatures for months and yet have a good performance and produce a fai amount of protein. The more fluorescent protein produced the better the visible signal CBD will provide.<br />
<br />
===Packaging===<br />
<br />
Being cell-free, Cell by Date, makes it easier to be embedded on food packaging. This calls for an efficient packaging method that is transparent (for the person to be able to see the fluorescent protein) but also well isolated from the actual food source (to avoid contamination). There are also 2 important factors that need to be taken into consideration. The first is the fact that the cell extract requires oxygen for the reaction to occur. What is not known is how much is needed. This raises the issue whether sealing CBD in a package with limited oxygen will prevent the reaction from occuring. The other factor is evaporation. The smaller the sample tested, the more the evaporation there was and the quicker the reaction mixture vanished. The packaging must therefore sustain the volume of the reaction for the lifespan of the food product.<br />
<br clear="all"><br />
[[Image:IC2007 CBD packaging.jpg|thumb|left|430px|'''CBD deactivated''' - Cold chain has not been disrupted]]<br />
[[Image:IC2007 CBD packaging2.jpg|thumb|left|430px|'''CBD activated''' (Red Glow) - Cold chain has been disrupted]]<br />
<br clear="all"><br />
<br clear="all"><br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Cell_by_Date/Testing << Testing ] | Conclusions | [https://2007.igem.org/Imperial Home >> ]</center></div>LucasCYhttp://2007.igem.org/wiki/index.php/File:IC2007_CBD_packaging2.jpgFile:IC2007 CBD packaging2.jpg2007-10-26T14:39:03Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/File:IC2007_CBD_packaging.jpgFile:IC2007 CBD packaging.jpg2007-10-26T14:38:20Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/User:LucasCYUser:LucasCY2007-10-26T04:01:42Z<p>LucasCY: </p>
<hr />
<div>I pity the fool who messes with my profile !<br />
<br />
[[Image:IC2007 blue.jpg]]</div>LucasCYhttp://2007.igem.org/wiki/index.php/File:IC2007_blue.jpgFile:IC2007 blue.jpg2007-10-26T04:00:29Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/User:Anthony_LUser:Anthony L2007-10-26T03:56:04Z<p>LucasCY: </p>
<hr />
<div>My never-ending love affairs ...<br />
<br />
[[Image:CrazyChucky.jpg]]<br />
<br />
[[Image:TheKing.jpg]]<br />
<br />
[[Image:Pronald.jpg]]<br />
<br />
[[Image:Hamburglar.jpg]]</div>LucasCYhttp://2007.igem.org/wiki/index.php/User:LucasCYUser:LucasCY2007-10-26T03:54:34Z<p>LucasCY: </p>
<hr />
<div></div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-25T15:44:20Z<p>LucasCY: /* Added control - Construct 2 */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
=Infector Detector: Future Work =<br />
<br><br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector detector is that it is not limited to just one infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. For example by using a construct that recognises AI-2<sup>[[#References |1]]</sup> , we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:IC2007 Conculsion4.jpg|thumb|right|390px|Tweaking sensitivity using LuxR]] <br />
The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br><br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity. Briefly, if we want to detect only highly progressed infections, we add little LuxR. If we want to detect infections with minimum progression, we saturate with AHL.<br><br><br />
Thus construct 2 can be used as a sensitivity attenuator.<br />
<br />
<br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged into either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as he can simply spray from a distance. The disadvantage being poor accuracy of application and more evaporation.<br />
<br />
<br />
A cream on the other hand will decrease significanlty any evaporation and will allow the user to apply the detector to specific areas of the catheter without the possibility of spraying the patient itself with detector. The disadvantage being here is the low diffusion rates of AHL and other compounds that need to cross the viscous cream to reach the actual detector assemblies. This might hinder rapid detection.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Testing << Testing] | Future Work | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-25T15:42:56Z<p>LucasCY: /* Packaging */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
=Infector Detector: Future Work =<br />
<br><br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector detector is that it is not limited to just one infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. For example by using a construct that recognises AI-2<sup>[[#References |1]]</sup> , we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:BB_c2.png|right|300px]]<br />
The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br><br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity. Briefly, if we want to detect only highly progressed infections, we add little LuxR. If we want to detect infections with minimum progression, we saturate with AHL.<br><br />
Thus construct 2 can be used as a sensitivity attenuator.<br />
<br />
[[Image:IC2007 Conculsion4.jpg|thumb|center|600px|Tweaking sensitivity using LuxR]] <br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged into either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as he can simply spray from a distance. The disadvantage being poor accuracy of application and more evaporation.<br />
<br />
<br />
A cream on the other hand will decrease significanlty any evaporation and will allow the user to apply the detector to specific areas of the catheter without the possibility of spraying the patient itself with detector. The disadvantage being here is the low diffusion rates of AHL and other compounds that need to cross the viscous cream to reach the actual detector assemblies. This might hinder rapid detection.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Testing << Testing] | Future Work | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-25T15:41:43Z<p>LucasCY: /* Battle a spectrum of infections */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
=Infector Detector: Future Work =<br />
<br><br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|left|]]<br />
The great potential of Infector detector is that it is not limited to just one infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. For example by using a construct that recognises AI-2<sup>[[#References |1]]</sup> , we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:BB_c2.png|right|300px]]<br />
The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br><br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity. Briefly, if we want to detect only highly progressed infections, we add little LuxR. If we want to detect infections with minimum progression, we saturate with AHL.<br><br />
Thus construct 2 can be used as a sensitivity attenuator.<br />
<br />
[[Image:IC2007 Conculsion4.jpg|thumb|center|600px|Tweaking sensitivity using LuxR]] <br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged into either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
<br />
<br />
<br />
<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as he can simply spray from a distance. The disadvantage being poor accuracy of application and more evaporation.<br />
<br clear="all"><br />
<br clear="all"><br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
<br />
<br />
A cream on the other hand will decrease significanlty any evaporation and will allow the user to apply the detector to specific areas of the catheter without the possibility of spraying the patient itself with detector. The disadvantage being here is the low diffusion rates of AHL and other compounds that need to cross the viscous cream to reach the actual detector assemblies. This might hinder rapid detection.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Testing << Testing] | Future Work | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-25T15:41:11Z<p>LucasCY: /* Battle a spectrum of infections */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Infector_Detector/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Infector_Detector/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
=Infector Detector: Future Work =<br />
<br><br />
==Battle a spectrum of infections==<br />
[[Image:IC2007 conclusion1.jpg|520px|center|]]<br />
The great potential of Infector detector is that it is not limited to just one infection. Adding sensitivity to AHL originating from biofilms is just the beginning. By tweaking the internal mechanisms of the construct, Infector Detector can be used to battle a range of catheter-related bacteremias. For example by using a construct that recognises AI-2<sup>[[#References |1]]</sup> , we can detect the presence of Klebsiella pneumoniae, a pathogenic bacterium ranked second to E. coli for urinary tract infections in older persons.<br />
<br><br />
<br clear="all"><br />
<br><br><br />
<br />
==Added control - Construct 2==<br />
<br><br />
[[Image:BB_c2.png|right|300px]]<br />
The main advantage of using construct 2 is that it provides an additional control mechanism for our detector meaning that you can tweak the detector sensitivity.<br><br />
Going into deeper detail, construct 1 can produce LuxR as soon as it is activated. LuxR's presence is necessary for the formation of AHL-LuxR complex and the subsequent activation of pLux (leading to GFP production). Construct 2 on the other hand does not have a LuxR producing part. It relies on the user to add the necessary LuxR to form the binding complex. This control over LuxR can thus act as a sort of attenuator to the sensitivity of Infector Detector.<br><br />
Having little LuxR present, will form very little binding complex with AHL and thus the sensitivity will decrease significantly. Saturating the detection compound with LuxR will maximise the sensitivity. Briefly, if we want to detect only highly progressed infections, we add little LuxR. If we want to detect infections with minimum progression, we saturate with AHL.<br><br />
Thus construct 2 can be used as a sensitivity attenuator.<br />
<br />
[[Image:IC2007 Conculsion4.jpg|thumb|center|600px|Tweaking sensitivity using LuxR]] <br />
<br />
<br clear="all"><br />
<br><br><br />
<br />
==Packaging==<br />
<br />
Infector Detector can be packaged into either a cream or a spray.<br />
<br>[[Image:IC2007 IDspray.jpg|thumb|150px|left|Infector Detector Spray]]<br />
<br />
<br />
<br />
<br />
A spray will provide easy application of the detector because it does not require the user to fiddle around with the urinary catheter as he can simply spray from a distance. The disadvantage being poor accuracy of application and more evaporation.<br />
<br clear="all"><br />
<br clear="all"><br />
[[Image:IC2007_IDPackaging.jpg|thumb|150px|right|Infector Detector Creme]]<br />
<br />
<br />
A cream on the other hand will decrease significanlty any evaporation and will allow the user to apply the detector to specific areas of the catheter without the possibility of spraying the patient itself with detector. The disadvantage being here is the low diffusion rates of AHL and other compounds that need to cross the viscous cream to reach the actual detector assemblies. This might hinder rapid detection.<br />
<br><br />
<br />
<br />
<br />
<br />
Both applications provide some advantages and disadvantages that must be weighed depending on the actual use scenario of Infector Detector in order to decide which is best.<br />
<br />
<br clear="all"><br />
<br clear="all"><br />
<br clear="all"><br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Testing << Testing] | Future Work | [https://2007.igem.org/Imperial Home >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Damien Balestrino et al. Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation. J Bacteriol. 2005 April; 187(8): 2870–2880.</div>LucasCYhttp://2007.igem.org/wiki/index.php/Imperial/Cell_by_Date/ConclusionImperial/Cell by Date/Conclusion2007-10-25T15:39:55Z<p>LucasCY: /* Extending life of reaction */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<html><br />
<link rel="stylesheet" href="/igem07/index.php?title=User:Dirkvs/Stylesheets/IC07persist.css&action=raw&ctype=text/css" type="text/css" /><br />
<br />
<div id="tabs"><br />
<ul><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Specification" title=""><span>Specifications</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Design" title=""><span>Design</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Modelling" title=""><span>Modelling</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Implementation" title=""><span>Implementation</span></a></li><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Testing" title=""><span>Testing</span></a></li><br />
<li><a class="current" href="https://2007.igem.org/Imperial/Cell_by_Date/Conclusion" title=""><span>Conclusion</span></a></li><br />
</ul><br />
</div><br />
<hr /><br />
<br clear="all"><br />
</html><br />
__NOTOC__<br />
= Cell by Date: Conclusion =<br />
<br />
Cell By Date is a great example of how synthetic biology can be integrated into our daily lives. When fully developed it can mimic the function of inducstrial level devices in the food industry at a fraction of the cost. Being a reporter of a break in the cold chain, CBD, can inform the end consumer whether the product he is buying has been exposed to abnormal storage conditions.<br />
<br />
This is best explained with an example. Imagine your local supermarket expecting a delivery of frozen lamb steaks. The delivery van arrives, the goods are unloaded and placed on the shelves awaiting for their first victim to arrive. Victim because what the supermarket manager and the end consumer are not aware of is that during their transport the van fridge broke down and thus the steaks were defrosted. The vab driver however fixed the fault, refroze the meat and told nothing upon his arrival to the supermarket. The whole incident will be kept under cover until the first cases of food poisoning arise from the comprimised lamb meat.<br />
<br />
This is were CBD comes in handy. By embedding a sticker of CBD on packaged goods, the consumer can check at any time whether the product quality has been comprimised by exposure to ubnormally high temperatures. Just a glance at the sticker and its bright fluorescent colour will inform the buyer of a potential risk. That is if the quality control expert of the supermarket does not spot it first.<br />
<br />
Even though not fully characterised, promising progress has been made and shows that even without tweaking the initial design, Cell by Date has a great potential just waiting to be exploited.<br />
<br />
= Future Work =<br />
<br />
===Extending the life of the reaction===<br />
<br />
A major constrain of CBD is its short lifetime. The cell-free extract used currently can only support it for a maximum of a few days or for the production of 12mg of protein whichever comes first. A good amount of research can go into developing cell extract that can be stored at various temperatures for months and yet have a good performance and produce a fai amount of protein. The more fluorescent protein produced the better the visible signal CBD will provide.<br />
<br />
===Packaging===<br />
<br />
Being cell-free, Cell by Date, makes it easier to be embedded on food packaging. This calls for an efficient packaging method that is transparent (for the person to be able to see the fluorescent protein) but also well isolated from the actual food source (to avoid contamination). There are also 2 important factors that need to be taken into consideration. The first is the fact that the cell extract requires oxygen for the reaction to occur. What is not known is how much is needed. This raises the issue whether sealing CBD in a package with limited oxygen will prevent the reaction from occuring. The other factor is evaporation. The smaller the sample tested, the more the evaporation there was and the quicker the reaction mixture vanished. The packaging must therefore sustain the volume of the reaction for the lifespan of the food product.<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Cell_by_Date/Testing << Testing ] | Future Work | [https://2007.igem.org/Imperial Home >> ]</center></div>LucasCY