http://2007.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Dirkvs&year=&month=2007.igem.org - User contributions [en]2024-03-28T09:26:37ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/File:CBD_steps.xlsFile:CBD steps.xls2007-10-27T01:09:48Z<p>Dirkvs: </p>
<hr />
<div></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_LabImperial/Wet Lab2007-10-27T00:20:37Z<p>Dirkvs: </p>
<hr />
<div>{{Template: IC07navmenu}}<br />
__NOTOC__<br />
<br clear="all"><br />
= Wet Lab Portal =<br />
Science is the study of all things physical, and central to the understanding of any particular phenomena is the process of experimentation and observation. In SynBio context, this is carried out as a method of verification and validation of design. <br />
<br />
While the engineering approach provides the practical application of scientific knowledge, it is also the platform by which we challenge our understanding and reasoning of biological concepts, where experimental observation serves to reinforce our old concepts, or acquire new knowledge. In other words, Science derives what Engineering derives from Science. And such is the dynamism of the exciting field of Synthetic Biology.<br />
<br />
The laboratory is the sanctuary of scientific experiment where rigorous testing can be conducted - and knowledge generated. The Wet Lab portal is an attempt to centralise all experimental designs and findings, and in here you will find all the methodology, as well as results of our findings. Welcome to our virtual Wet Lab!<br />
<br />
<br><br />
{| cellpadding="0" border="0" align="left"<br />
|-<br />
| [[Imperial/Wet_Lab/Lab_Notebook|<font face="Arial" size=3 style="color:#000099">'''Lab Notebook'''</font>]]<br /> <font size=2>Daily Record of Wet Lab</font><br />
<br><br />
|-<br />
| [[Imperial/Wet_Lab/Protocols|<font face="Arial" size=3 style="color:#000099">'''Protocols'''</font>]]<br /> <font size=2>General & Testing Protocols</font><br />
<br><br />
|-<br />
| [[Imperial/Wet_Lab/DNA_Constructs|<font face="Arial" size=3 style="color:#000099">'''DNA Constructs'''</font>]]<br /> <font size=2>Overview of DNA Constructs</font><br />
|}<br />
<br clear="all"><br />
<br />
<br />
<center>| [[Imperial | Home >>]]</center></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_LabImperial/Wet Lab2007-10-27T00:19:22Z<p>Dirkvs: /* Wet Lab Portal */</p>
<hr />
<div>{{Template: IC07navmenu}}<br />
__NOTOC__<br />
<br />
= Wet Lab Portal =<br />
Science is the study of all things physical, and central to the understanding of any particular phenomena is the process of experimentation and observation. In SynBio context, this is carried out as a method of verification and validation of design. <br />
<br />
While the engineering approach provides the practical application of scientific knowledge, it is also the platform by which we challenge our understanding and reasoning of biological concepts, where experimental observation serves to reinforce our old concepts, or acquire new knowledge. In other words, Science derives what Engineering derives from Science. And such is the dynamism of the exciting field of Synthetic Biology.<br />
<br />
The laboratory is the sanctuary of scientific experiment where rigorous testing can be conducted - and knowledge generated. The Wet Lab portal is an attempt to centralise all experimental designs and findings, and in here you will find all the methodology, as well as results of our findings. Welcome to our virtual Wet Lab!<br />
<br />
<br><br />
{| cellpadding="0" border="0" align="left"<br />
|-<br />
| [[Imperial/Wet_Lab/Lab_Notebook|<font face="Arial" size=3 style="color:#000099">'''Lab Notebook'''</font>]]<br /> <font size=2>Daily Record of Wet Lab</font><br />
<br><br />
|-<br />
| [[Imperial/Wet_Lab/Protocols|<font face="Arial" size=3 style="color:#000099">'''Protocols'''</font>]]<br /> <font size=2>General & Testing Protocols</font><br />
<br><br />
|-<br />
| [[Imperial/Wet_Lab/DNA_Constructs|<font face="Arial" size=3 style="color:#000099">'''DNA Constructs'''</font>]]<br /> <font size=2>Overview of DNA Constructs</font><br />
|}<br />
<br clear="all"><br />
<br />
<br />
<center>| [[Imperial | Home >>]]</center></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/FunImperial/Fun2007-10-26T23:49:17Z<p>Dirkvs: /* Infamous Quotes */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
__NOTOC__<br />
<br />
= Fun with iGEM =<br />
While the iGEM ride has its ups and downs, we took every oppurtunity to safekeep the good things that kept us going - the fun bits!<br />
<br />
<center><br />
==== We're on UK Television!! - Channel 4 Interview at the Freemont Lab ====<br />
<html><br />
<object width="425" height="355"><param name="movie" value="http://www.youtube.com/v/A-mCWIGVgmQ&rel=1"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/A-mCWIGVgmQ&rel=1" type="application/x-shockwave-flash" wmode="transparent" width="425" height="355"></embed></object><br />
</html></center><br />
<br clear="all"><br />
<br><br />
<br />
<br />
<br />
== Pictures ==<br />
{|border="0" width="90%" align="center"<br />
|-<br />
|width="50%"| <br />
'''Snapshots'''<br />
<br />
<html><a href="http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Entertainment/Pictures"> <br />
<img src="https://static.igem.org/mediawiki/2007/8/85/IC07_teamsnap.JPG" width="400px"><br />
<br />
<b>Proof of the iGEM Experience!</b></a></html><br />
<br />
|width="50%"| <br />
'''The Whiteboard'''<br />
<br />
<html><a href="http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Entertainment/Whiteboard"> <br />
<img src="https://static.igem.org/mediawiki/2007/0/0d/IC07_moodchart.JPG" width="400px"><br />
<br />
<b>The Everchanging Face of the Whiteboard</b></a></html><br />
|}<br />
<br clear="all"><br />
<br />
<br />
== Infamous Quotes ==<br />
*"Clear wiki, Clear mind" --- Matthieu<br />
<br />
*"I can't smell my fingers... *snif snif* ...and answer your question."--- Lucas<br />
<br />
*"For instance..."--- Jerry<br />
<br />
*"Lucas! Can you give me one night! *hearts*" --- Ben<br />
<br />
*"For instance..."--- Jerry<br />
<br />
*"Oh no you don't..."--- James<br />
<br />
*"For instance..."--- Jerry<br />
<br />
*"Hmm."--- Cheuk ka<br />
<br />
*"GFP says... no Alex says, its takes 4-8 hours"--- Ben<br />
<br />
*"Jerry you are needed in the lab! you are the only one who can label "TL"! *giggles*"--- Ben<br />
<br />
*Spice Girls <br />
**Baby Spice- PPx<br />
**Ginger Spice- Jerry<br />
**Posh Spice- Ben<br />
**Scary Spice- <br />
**Sporty Spice-<br />
<br />
*"I am chewing gum."--- Lucas<br />
<br />
*"The ignorant will never be guilty"--- Mistress(BOSS) Peixuan Pey<br />
<br />
*"My surname is KHAN."--- Maira Khan<br />
<br />
*"I am not a gentleman. I am a lady!"--- Lucas<br />
<br />
*"What is after F...G?"--- Lucas<br />
<br />
*"I like Alex."--- James<br />
<br />
*"I am not that gay YET."--- Ben<br />
<br />
*"You have the British face.... I like..."--- Lucas to James<br />
<br />
*"It is too painful." -- Vincent, on repeating the line "I'm quite impressed".<br />
<br />
<br />
<br />
<center>| [[Imperial |Back to Home]]</center></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/FunImperial/Fun2007-10-26T23:48:57Z<p>Dirkvs: /* Infamous Quotes */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
__NOTOC__<br />
<br />
= Fun with iGEM =<br />
While the iGEM ride has its ups and downs, we took every oppurtunity to safekeep the good things that kept us going - the fun bits!<br />
<br />
<center><br />
==== We're on UK Television!! - Channel 4 Interview at the Freemont Lab ====<br />
<html><br />
<object width="425" height="355"><param name="movie" value="http://www.youtube.com/v/A-mCWIGVgmQ&rel=1"></param><param name="wmode" value="transparent"></param><embed src="http://www.youtube.com/v/A-mCWIGVgmQ&rel=1" type="application/x-shockwave-flash" wmode="transparent" width="425" height="355"></embed></object><br />
</html></center><br />
<br clear="all"><br />
<br><br />
<br />
<br />
<br />
== Pictures ==<br />
{|border="0" width="90%" align="center"<br />
|-<br />
|width="50%"| <br />
'''Snapshots'''<br />
<br />
<html><a href="http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Entertainment/Pictures"> <br />
<img src="https://static.igem.org/mediawiki/2007/8/85/IC07_teamsnap.JPG" width="400px"><br />
<br />
<b>Proof of the iGEM Experience!</b></a></html><br />
<br />
|width="50%"| <br />
'''The Whiteboard'''<br />
<br />
<html><a href="http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Entertainment/Whiteboard"> <br />
<img src="https://static.igem.org/mediawiki/2007/0/0d/IC07_moodchart.JPG" width="400px"><br />
<br />
<b>The Everchanging Face of the Whiteboard</b></a></html><br />
|}<br />
<br clear="all"><br />
<br />
<br />
== Infamous Quotes ==<br />
*"Clear wiki, Clear mind" --- Matthieu<br />
<br />
*"I can't smell my fingers... *snif snif* and answer your question."--- Lucas<br />
<br />
*"For instance..."--- Jerry<br />
<br />
*"Lucas! Can you give me one night! *hearts*" --- Ben<br />
<br />
*"For instance..."--- Jerry<br />
<br />
*"Oh no you don't..."--- James<br />
<br />
*"For instance..."--- Jerry<br />
<br />
*"Hmm."--- Cheuk ka<br />
<br />
*"GFP says... no Alex says, its takes 4-8 hours"--- Ben<br />
<br />
*"Jerry you are needed in the lab! you are the only one who can label "TL"! *giggles*"--- Ben<br />
<br />
*Spice Girls <br />
**Baby Spice- PPx<br />
**Ginger Spice- Jerry<br />
**Posh Spice- Ben<br />
**Scary Spice- <br />
**Sporty Spice-<br />
<br />
*"I am chewing gum."--- Lucas<br />
<br />
*"The ignorant will never be guilty"--- Mistress(BOSS) Peixuan Pey<br />
<br />
*"My surname is KHAN."--- Maira Khan<br />
<br />
*"I am not a gentleman. I am a lady!"--- Lucas<br />
<br />
*"What is after F...G?"--- Lucas<br />
<br />
*"I like Alex."--- James<br />
<br />
*"I am not that gay YET."--- Ben<br />
<br />
*"You have the British face.... I like..."--- Lucas to James<br />
<br />
*"It is too painful." -- Vincent, on repeating the line "I'm quite impressed".<br />
<br />
<br />
<br />
<center>| [[Imperial |Back to Home]]</center></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:45:12Z<p>Dirkvs: /* 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 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 />
<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 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 a construct that recognises AI-2<sup>[[#References |1]]</sup>, for example, 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 the detection compounds through a viscous cream is lower. This will slow down the response of the system.<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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:40:13Z<p>Dirkvs: /* 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 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 />
<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 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 a construct that recognises AI-2<sup>[[#References |1]]</sup>, for example, 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:40:00Z<p>Dirkvs: /* 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 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 />
<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 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 a construct that recognises AI-2<sup>[[#References |1]]</sup>, for example, 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:39:44Z<p>Dirkvs: /* 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 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 />
<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 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 a construct that recognises AI-2<sup>[[#References |1]]</sup>, for example, 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.<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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:37:58Z<p>Dirkvs: /* 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 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 />
<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 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 a construct that recognises AI-2<sup>[[#References |1]]</sup>, for example, 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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:36:40Z<p>Dirkvs: /* 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 />
<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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:35:48Z<p>Dirkvs: /* 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 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"|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 />
<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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:35:07Z<p>Dirkvs: /* 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 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"|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 />
<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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:34:26Z<p>Dirkvs: /* 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 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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:34:08Z<p>Dirkvs: /* Infector Detector: Conclusion */</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 />
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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 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>'''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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:32:00Z<p>Dirkvs: /* 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 />
*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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ConclusionImperial/Infector Detector/Conclusion2007-10-26T23:31:22Z<p>Dirkvs: /* 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 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 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 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 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>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Results/Res1.3/Converting_UnitsImperial/Wet Lab/Results/Res1.3/Converting Units2007-10-26T23:29:15Z<p>Dirkvs: /* Conversion of Data */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
<br clear="all"><br />
=Conversion of Data=<br />
<br />
Using our calibration cuvre of GFPmut3b in our ''in vitro'' chassis we are able to convert the fluorescence units into a number of different units. We have looked at how we can convert between four different units:<br />
<br />
# Fluorescence to Concentration of GFPmut3b<br />
# Fluorescence to Moles of GFPmut3b<br />
# Fluorescence to Molecules of GFPmut3b<br />
# Fluorescence to Molecules of GFPmut3b per plasmid<br />
<br />
====Advantages====<br />
There is a massive advantage for being able to interconvert between units. As of yet there is no standardised units for ''in vitro'', for ''in vivo'' work on the F2620 used the standard units <font color=green>'''molecules of GFPmut3b synthesised per second per cfu (colony forming units)'''</font>. The units above allow a variety of different units to be used including a normalised unit based upon the number of DNA plasmids present in the chassis.<br />
<br />
====Constraints====<br />
There are certain constraints to using our calibration curve:<br />
*First the total volume must be kept constant as volume can affect fluorescence, we chose a volume of 60&micro;l. <br />
*The type of cell extract may also be important, this is for Promega S30 Cell Extract<br />
*The DNA concentration used for the calibration curve could affect the fluorescence measure, therefore we standardised the DNA used in our experiments to 4&micro;g. <br />
*Finally because of fluorometer variation then at least the same conditions must be used if not the same fluorometer.<br />
<br />
The diagram below shows the various conversions that can be carried out:<br />
<br />
<br />
{|align="left"<br />
| width="500px"|<br>[[Image:Conversion Units.PNG|thumb|500px|]] <br />
|}</div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Results/ID3.1Imperial/Wet Lab/Results/ID3.12007-10-26T23:28:30Z<p>Dirkvs: /* ''In vitro'' Testing of pTet-LuxR-pLux-GFPmut3b Construct, varying concentrations of AHL */</p>
<hr />
<div>{{Template: IC07navmenu}}<br />
__NOTOC__<br />
<br />
= ''In vitro'' Testing of pTet-LuxR-pLux-GFPmut3b Construct with varying concentrations of AHL=<br />
<br />
==Aims==<br />
To characterise the [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] construct ''in vitro''. The characterisation is based upon giving inputs of varying AHL concentration and measuring the fluorescent output.<br />
<br />
<br />
==Materials and Methods==<br />
Link to the [[Imperial/Wet_Lab/Protocols/ID3.1|Protocols]]<br />
<br />
==Results==<br />
{|align="center"<br />
| width="600px"|<br>[[Image:IC 2007 Fina AHL.PNG|thumb|600px|Figure 1.1 '''pTet-LuxR-pLux-GFPmut3b''' ''in vitro'' - The graph shows the concentration of GFPmut3b against time for varying levels of AHL]] <br />
|}<br />
{|align="center"<br />
| width="600px"|<br>[[Image:IC 2007 Fugure 1.2 plasmid.PNG|thumb|600px|Figure 1.2:pTet-LuxR-pLux-GFPmut3b ''in vitro'' - The graph shows the molecules of GFPmut3b synthesised per plasmid within the ''in vitro''chassis against time for varying levels of AHL]] <br />
|}<br />
<br />
<br />
<br />
'''Controls:'''<br />
*Negative Control- '''pTet-LuxR-pLux-GFPmut3b''' with no AHL<br />
*Negative Control.2.- '''pLux''' with 1000nM AHL<br />
<br />
<br />
'''Constants:'''<br />
*Temperature - 25&deg;C<br />
*Volume - 60ul<br />
*Sampling on same plate and every 30 minutes<br />
<br />
'''Raw Data'''<br />
<br />
==Discussion==<br />
The results from the experiment were of fluorescence against time for varying levels of AHL. Using our calibration curves we managed to convert the fluorescence into concentration of GFPmut3b produced (figure 1.1) and also into Molecules of GFPmut3b synthesised per plasmid (figure 1.2). See the how the calibration curves allowed us to do this by following this link to conversion of units page (LINK).<br />
<br />
The idea behind using molecules of GFPmut3b is to try to standardise our units for the chassis and normalise our data based upon the number of DNA plasmids present in our system. This idea is similar to that for the F2620 which uses the units GFP molecules synthesized per second per cell. Trying to normalise this data with the plasmids helps to make our data independent of the DNA used in this assay, i.e. for other constructs tested and characterized in vitro.<br />
<br />
There are several key parameters that can be extracted from the data. It looks as if our system becomes saturated to AHL ( the input) above 100nM of AHL, it can be seen that there is little difference between 1000nM and 100nM. If we assume the GFP synthesis 1000nM to be the maximum then the switch point , defined when the output is 50% of the max, is around 20nM of AHL.<br />
<br />
The response time is a key characteristic however from this data it is difficult to get an accurate value. The problem is that because the temperature was regulated sampling had to be restricted to every 30 minutes, meaning our best approximation is <30minutes.<br />
<br />
The sensitivity of the chassis and construct can also be deduced for the range of AHL concentrations that we looked at. The sensitivity to AHL is <5nM.<br />
<br />
<br />
<br />
==Conclusion==<br />
These are the characteristics we have extracted<br />
<br />
Switch Point= ~20nM AHL<br><br />
Response Time= <30 minutes<br><br />
Sensitivity = <5nM AHL<BR><br />
<br />
In terms of the specification of the Infector Detector Application the chassis and construct is sensitive enough.</div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Results/ID3.1Imperial/Wet Lab/Results/ID3.12007-10-26T23:28:08Z<p>Dirkvs: /* ''In vitro'' Testing of pTet-LuxR-pLux-GFPmut3b Construct with varying concentrations of AHL */</p>
<hr />
<div>{{Template: IC07navmenu}}<br />
__NOTOC__<br />
<br />
= ''In vitro'' Testing of pTet-LuxR-pLux-GFPmut3b Construct, varying concentrations of AHL=<br />
<br />
==Aims==<br />
To characterise the [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] construct ''in vitro''. The characterisation is based upon giving inputs of varying AHL concentration and measuring the fluorescent output.<br />
<br />
<br />
==Materials and Methods==<br />
Link to the [[Imperial/Wet_Lab/Protocols/ID3.1|Protocols]]<br />
<br />
==Results==<br />
{|align="center"<br />
| width="600px"|<br>[[Image:IC 2007 Fina AHL.PNG|thumb|600px|Figure 1.1 '''pTet-LuxR-pLux-GFPmut3b''' ''in vitro'' - The graph shows the concentration of GFPmut3b against time for varying levels of AHL]] <br />
|}<br />
{|align="center"<br />
| width="600px"|<br>[[Image:IC 2007 Fugure 1.2 plasmid.PNG|thumb|600px|Figure 1.2:pTet-LuxR-pLux-GFPmut3b ''in vitro'' - The graph shows the molecules of GFPmut3b synthesised per plasmid within the ''in vitro''chassis against time for varying levels of AHL]] <br />
|}<br />
<br />
<br />
<br />
'''Controls:'''<br />
*Negative Control- '''pTet-LuxR-pLux-GFPmut3b''' with no AHL<br />
*Negative Control.2.- '''pLux''' with 1000nM AHL<br />
<br />
<br />
'''Constants:'''<br />
*Temperature - 25&deg;C<br />
*Volume - 60ul<br />
*Sampling on same plate and every 30 minutes<br />
<br />
'''Raw Data'''<br />
<br />
==Discussion==<br />
The results from the experiment were of fluorescence against time for varying levels of AHL. Using our calibration curves we managed to convert the fluorescence into concentration of GFPmut3b produced (figure 1.1) and also into Molecules of GFPmut3b synthesised per plasmid (figure 1.2). See the how the calibration curves allowed us to do this by following this link to conversion of units page (LINK).<br />
<br />
The idea behind using molecules of GFPmut3b is to try to standardise our units for the chassis and normalise our data based upon the number of DNA plasmids present in our system. This idea is similar to that for the F2620 which uses the units GFP molecules synthesized per second per cell. Trying to normalise this data with the plasmids helps to make our data independent of the DNA used in this assay, i.e. for other constructs tested and characterized in vitro.<br />
<br />
There are several key parameters that can be extracted from the data. It looks as if our system becomes saturated to AHL ( the input) above 100nM of AHL, it can be seen that there is little difference between 1000nM and 100nM. If we assume the GFP synthesis 1000nM to be the maximum then the switch point , defined when the output is 50% of the max, is around 20nM of AHL.<br />
<br />
The response time is a key characteristic however from this data it is difficult to get an accurate value. The problem is that because the temperature was regulated sampling had to be restricted to every 30 minutes, meaning our best approximation is <30minutes.<br />
<br />
The sensitivity of the chassis and construct can also be deduced for the range of AHL concentrations that we looked at. The sensitivity to AHL is <5nM.<br />
<br />
<br />
<br />
==Conclusion==<br />
These are the characteristics we have extracted<br />
<br />
Switch Point= ~20nM AHL<br><br />
Response Time= <30 minutes<br><br />
Sensitivity = <5nM AHL<BR><br />
<br />
In terms of the specification of the Infector Detector Application the chassis and construct is sensitive enough.</div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ModellingImperial/Infector Detector/Modelling2007-10-26T23:25:17Z<p>Dirkvs: /* Implementation & Reaction Network */</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 class="current" 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 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: Modelling =<br />
<br />
==Introduction==<br />
Infector Detector (ID) is a simple biological detector designed to expose the presence of a bacterial biofilm. It functions by exploiting the inherent AHL (Acetyl Homoserine Lactone) production employed by certain types of quorum-sensing bacteria, in the formation of such structures. The [[Imperial/Infector_Detector/Design| design]] phase of our project has yielded two possible system constructs.<br />
<br />
==Implementation & Reaction Network==<br />
<br />
In line with the concept of abstraction in Synthetic Biology, the correlation of the output of the proposed system constructs to their inputs, can be visualized by the following black-box illustrations of the two cases. <br />
It is evident that AHL is an input to both constructs; a function of the particular biofilm. Furthermore, energy and promoter concentration are included as auxillary inputs to both system constructs. LuxR, is an additional input, exclusive to construct 2, which lacks constitutive expression of LuxR by pTET.<br><br />
(this of course occuring within our cell-free chassis)<br />
{|-<br />
|[[Image: BB c1.png|thumb|440px|'''Figure 1''': Black-box for Construct 1]]||[[Image: BB c2.png|thumb|440px|'''Figure 2''': Black-box for Construct 2]]<br />
|}<br />
<br />
====''The Reaction Network''====<br />
<br />
Both designs are based on the following reaction network:<br />
<br />
*AHL is assumed to diffuse freely "into" the system (we are dealing with a cell-free system, which comes into direct contact with the biofilm). <br />
*The target AHL molecule binds with the monomeric protein LuxR.<br />
*LuxR is either constitutively produced by construct 1, or directly introduced in purified form, as part of construct 2.<br />
*The binding of these two proteins yields the intermediating LuxR-AHL complex, A. We call k<sub>2</sub> and k<sub>3</sub> the kinetic constants of the forward and backward reactions respectively. <br />
*The formed transcription factor activates the transcription of the pLux operon, which codes for the relevant reporter protein, GFP. Activation occurs by way of the reversible binding of this transcription factor, A, to the response sequences in the operon (k<sub>4</sub> and k<sub>5</sub>) <br />
*This leads to recruitment of RNA polymerase and increases the frequency of transcription initiation (Fuqua et al., 2001) of the construct gfp gene (strictly forward reaction, governed by k<sub>6</sub>).<br />
<br />
{|-<br />
|[[Image: IC07 Const1.png|thumb|440px|'''Figure 3''': Reaction network for Construct 1 (Energy-dependent)]] || [[Image: IC07 C2.png|thumb|440px|'''Figure 4''': Reaction network for Construct 2 (Energy-dependent)]] <br />
|}<br />
<br clear="all"><br />
<br />
==Representative Model==<br />
<br />
In developing this model, we were interested in the behaviour at steady-state, that is when the system has equilibrated and the concentrations of the state variables remain constant.<br />
<br />
'''A Resource-Dependent Model'''<br />
<br />
To simulate the behaviour of both constructs we have developed an ordinary differential equations (ODE) system that describes the evolution with time of the concentrations of the molecules involved in the reaction network. <br><br />
At reasonably high molecular concentrations of the state variables, such a model can be adopted instead of the more accurate stochastic model without any risk of major error. The advantages of the ODE approach in term of complexity and computing time/power are non negligible.<br />
<br>Our model depends not only on the reaction network described above but also on the following considerations:<br />
<br><br />
*The only difference is with regards to the parameter k<sub>1</sub>, the maximum transcription rate of the constitutive promoter (pTET). Therefore in construct 1, k<sub>1</sub> is non-zero; k<sub>1</sub> = 0 for construct 2 (which lacks pTET).<br />
*The chassis analysis conducted in the cell-free section (wiki link) has shown that some resource – dependent term had to be introduced to curb the synthesis of protein in a cell-free system. We retained the same curbing function as with the chassis characterisation.<br />
*In theory the cost of the synthesis of a protein is proportional to the length of the coding region. Since GFP and LuxR have coding regions of roughly same lengths (800-900 base pairs) we assume an equal cost for both proteins.<br />
*We assume no cooperativity in any of the bindings. <br />
<br />
'''The Equations'''<br />
<br />
[[Image:IC07 Model2.png|thumb|center|600px|'''Model 2''', an energy-dependent network, where the dependence on energy assumes Hill-like dynamics]]<br />
<br clear="all"><br />
<br />
where [E] represents the [nutrient] or ["energy"] within the system. The energy dependence is assumed to follow Hill-like Dynamics. <br />
<br />
<br />
The parameters of our model are described in the table below<br />
<br />
====''Model Parameters''====<br />
<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Parameter <br />
! Description<br />
|-<br />
|<font color = blue>''Kinetic <br> Constants'' </font><br />
|<br />
|-<br />
| k<sub>1</sub><br />
| Maximal constitutive transcription of LuxR by pTET<br />
|-<br />
|k<sub>2</sub><br />
|Binding between LuxR and AHL<br />
|-<br />
|k<sub>3</sub><br />
|Dissociation of protein complex LuxR-AHL (A)<br />
|-<br />
|k<sub>4</sub><br />
|Binding between A and pLux promoter<br />
|-<br />
|k<sub>5</sub><br />
|Dissociaton of A-pLux complex<br />
|-<br />
|k<sub>6</sub><br />
|Transcription of GFP - k<sub>6</sub> proportional to amount of DNA in solution<br />
|-<br />
|<font color = blue>''Degradation <br> Rates'' </font><br />
|<br />
|-<br />
|&delta;<sub>LuxR</sub><br />
|Degradation rate of LuxR<br><br />
&delta;<sub>LuxR</sub> is negligible – assumed to be zero<br />
|-<br />
|&delta;<sub>AHL</sub><br />
|Degradation rate of AHL<br><br />
&delta;<sub>AHL</sub> is negligible – assumed to be zero<br />
|-<br />
|&delta;<sub>GFP</sub><br />
|Degradation rate of GFP<br><br />
&delta;<sub>GFP</sub> is negligible – assumed to be zero<br />
|-<br />
|<font color = blue>''Hill Co-operativity''</font><br />
|<br />
|-<br />
|n<br />
|Co-operativity coefficient describing the degree <br>of energy dependence, which follows Hill-like dynamics<br />
|-<br />
|<font color = blue>''Energy consumption <br> of transcription''</font> <br />
|<br />
|-<br />
|&alpha;<sub>1</sub><br />
|Energy consumption due to constitutive transcription of LuxR<br />
|-<br />
|&alpha;<sub>2</sub><br />
|Energy consumption due to transcription of ''gfp'' gene<br />
|-<br />
|<font color = blue>''Initial Conditions''</font> <br />
|<br />
|-<br />
|P<sub>0</sub><br />
|Initial Concentration of Promoters <br><br />
P<sub>0</sub> represents the amount of DNA inserted into the extract<br />
|-<br />
|LuxR<sub>0</sub><br />
|Initial Concentration of LuxR<br />
<br />
|}<br />
<br />
<br><br />
<br />
We now present the essential features of the system behaviour, simulated for a given set of parameters.<br />
<br />
==Simulations==<br />
<br />
Presented below are the most essential results of the simulations performed. <br />
<br />
In light of the rapid equilibrium approximation which was employed in developing the model, relatively high k-values were selected: <br />
<br>k2 = k3 = k4 = k5 = 100 (dynamical equilibrium); k1 = 0.3 (constitutive transcription by pTet); k6 = 20*k1 (transcription of ''gfp'');<br />
<br />
<br />
<br />
==== 1. Construct 1: [GFP] vs time - varying [AHL]<sub>0</sub>====<br />
<br />
[[Image: C1 AHL GFP.png|thumb|left|430px|'''Figure 5''': Investigation of the effect on GFP expression by <br>'''Construct 1''', when the initial [AHL] is varied i.e. AHL<sub>0</sub> = 0.1, 1, 5, 10, 50 & 100 a.u. (arbitrary units)]]<br />
<br />
[[Image: C1 AHL Energy.png|thumb|right|430px|'''Figure 6''': Comparison of energy consumption of '''Construct''' 1, when the initial [AHL] is varied i.e. AHL<sub>0</sub> = 0.1, 1, 5, 10, 50 & 100 a.u (arbitrary units)]]<br />
<br />
====Discussion====<br />
<br />
Figures 5 and 6 illustrate GFP expression and Energy depletion of construct 1, at various initial AHL concentrations.<br />
<br />
The absolute value of the peak expression is a function of the various rate constants. Here our analysis serves to illustrate the general behaviour offered by construct 1 – a qualitative approach.<br />
<br />
As initial [AHL] is increased, the level of expression increases accordingly to a point were there is negligible difference between the maximal outputs between adjacent tested cases of [AHL]. In fact, from this figure, and for this set of parameters, it is suggestive that saturation occurs at approximately 5 a.u.; in fact, the difference in maximal GFP output between when AHL is increased a several-fold is less than 10%. <br />
<br />
Figure 6, the energy depletion plot, serves to illustrate the effect of increased initial concentrations of AHL on consumption of energy. More resources (promoters) need to be employed to "accommodate" the increasing [AHL]. This obviously increases the rate of energy depletion, until it levels off, as saturating behaviour has been attained - promoter saturation. This is the likely explanation for the "saturation curve" obtained from the experimental data, since the protein degradation terms themselves are almost negligible.<br />
<br />
====2. Construct 1: [GFP] vs [AHL] - transfer function curve====<br />
<br />
{|-<br />
| [[Image: C1 transfer 300min.png|thumb|left|430px|'''Figure 7''': Transfer function of construct 1, after t = 300 min(x-axis is log-scaled). [AHL] measured in arbitrary units]] || [[Image: C1 Transfer 1000min.png|thumb|right|430px|'''Figure 8''': Transfer function of construct 1, after t = 1000min (x-axis is log-scaled) [AHL] measured in arbitrary units]]<br />
|}<br />
<br clear="all"><br />
<br />
====Discussion====<br />
Figure 7 serves to amplify the essential claims from the previous simulation. Saturative behaviour occurs at approximately [AHL] = 5-10 arbitrary units (a.u.). The effect of increasing initial [AHL] beyond this level is of negligible effect on [GFP] expression, for initially available resources. This first transfer function is taken at t=300min. <br />
We would expect the curve to be considerably reduced at a later instance. This is due to reduced energy resources. This ties in with what is observed in figure 8, at t = 1000 above: significantly reduced peak expression of GFP.<br />
<br />
====Simulation 3 – effect of varying GFP degradation term on dynamic behaviour of construct 1====<br />
[[Image: C1 dGFP.png|thumb|left|450px|'''Figure 9''': ]]<br clear="all"><br />
<br />
====Discussion====<br />
This simulation illustrates the effect of delta_GFP on the expression of GFP by construct 1. At small, delta_GFP, the steady-state approach clearly can be attributed to the considerable rate of energy depletion. When the degradation terms are more significant, their effect is considerably greater than the effect of limited resources.<br />
If our model were not energy-dependent, then the simulation for &delta;<sub>GFP</sub> tending to zero should yield positively sloped parabolic curve.<br />
<br />
=== Conclusions ===<br />
<br />
From the initial simulations performed, it was suggested that construct 2 was more effective than construct 1 on the basis of sensitivity, response time and maximal output of reporter. Construct 1 was superior in terms of energy-efficiency. Its life-time is far greater than that of its counterpart. <br />
<br />
Experimental work was performed on both constructs. The notable feature of these endeavours was that Construct 2 was eventually aborted due to difficulty in effectively purifying LuxR. Since our initial simulations are promising with regards to this device, it should be investigated more fully in the future.<br />
In any case, the simulations for construct 1, tie in with that presented by the experimental team, and are a fullfilment of our specifications.<br />
<br />
===Software===<br />
<br />
All deterministic simulations were performed using Matlab 7 (The MathWorks Inc., Natick, MA).<br />
*m-files of all simulations are available on our [https://2007.igem.org/Imperial/Dry_Lab/Software Software] page.<br />
<br />
===References===<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Design << Design] | Modelling | [https://2007.igem.org/Imperial/Infector_Detector/Implementation Implementation >>]<br />
</center></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Infector_Detector/ModellingImperial/Infector Detector/Modelling2007-10-26T23:19:35Z<p>Dirkvs: /* 2. Construct 1: [GFP] vs [AHL] - transfer function curve */</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 class="current" 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 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: Modelling =<br />
<br />
==Introduction==<br />
Infector Detector (ID) is a simple biological detector designed to expose the presence of a bacterial biofilm. It functions by exploiting the inherent AHL (Acetyl Homoserine Lactone) production employed by certain types of quorum-sensing bacteria, in the formation of such structures. The [[Imperial/Infector_Detector/Design| design]] phase of our project has yielded two possible system constructs.<br />
<br />
==Implementation & Reaction Network==<br />
<br />
In line with the concept of abstraction in Synthetic Biology, the correlation of the output of the proposed system constructs to their inputs, can be visualized by the following black-box illustrations of the two cases. <br />
It is evident that AHL is an input to both constructs; a function of the particular biofilm. Furthermore, energy and promoter concentration are included as auxillary inputs to both system constructs. LuxR, is an additional input, exclusive to construct 2, which lacks constitutive expression of LuxR by pTET.<br><br />
(this of course occuring within our cell-free chassis)<br />
<br />
[[Image: BB c1.png|thumb|left|440px|'''Figure 1''': Black-box for Construct 1]]<br />
[[Image: BB c2.png|thumb|right|440px|'''Figure 2''': Black-box for Construct 2]]<br />
<br />
====''The Reaction Network''====<br />
<br />
Both designs are based on the following reaction network:<br />
<br />
*AHL is assumed to diffuse freely "into" the system (we are dealing with a cell-free system, which comes into direct contact with the biofilm). <br />
*The target AHL molecule binds with the monomeric protein LuxR.<br />
*LuxR is either constitutively produced by construct 1, or directly introduced in purified form, as part of construct 2.<br />
*The binding of these two proteins yields the intermediating LuxR-AHL complex, A. We call k<sub>2</sub> and k<sub>3</sub> the kinetic constants of the forward and backward reactions respectively. <br />
*The formed transcription factor activates the transcription of the pLux operon, which codes for the relevant reporter protein, GFP. Activation occurs by way of the reversible binding of this transcription factor, A, to the response sequences in the operon (k<sub>4</sub> and k<sub>5</sub>) <br />
*This leads to recruitment of RNA polymerase and increases the frequency of transcription initiation (Fuqua et al., 2001) of the construct gfp gene (strictly forward reaction, governed by k<sub>6</sub>). <br />
[[Image: IC07 Const1.png|thumb|left|440px|'''Figure 3''': Reaction network for Construct 1 (Energy-dependent)]]<br />
[[Image: IC07 C2.png|thumb|right|440px|'''Figure 4''': Reaction network for Construct 2 (Energy-dependent)]] <br />
<br clear="all"><br />
<br />
==Representative Model==<br />
<br />
In developing this model, we were interested in the behaviour at steady-state, that is when the system has equilibrated and the concentrations of the state variables remain constant.<br />
<br />
'''A Resource-Dependent Model'''<br />
<br />
To simulate the behaviour of both constructs we have developed an ordinary differential equations (ODE) system that describes the evolution with time of the concentrations of the molecules involved in the reaction network. <br><br />
At reasonably high molecular concentrations of the state variables, such a model can be adopted instead of the more accurate stochastic model without any risk of major error. The advantages of the ODE approach in term of complexity and computing time/power are non negligible.<br />
<br>Our model depends not only on the reaction network described above but also on the following considerations:<br />
<br><br />
*The only difference is with regards to the parameter k<sub>1</sub>, the maximum transcription rate of the constitutive promoter (pTET). Therefore in construct 1, k<sub>1</sub> is non-zero; k<sub>1</sub> = 0 for construct 2 (which lacks pTET).<br />
*The chassis analysis conducted in the cell-free section (wiki link) has shown that some resource – dependent term had to be introduced to curb the synthesis of protein in a cell-free system. We retained the same curbing function as with the chassis characterisation.<br />
*In theory the cost of the synthesis of a protein is proportional to the length of the coding region. Since GFP and LuxR have coding regions of roughly same lengths (800-900 base pairs) we assume an equal cost for both proteins.<br />
*We assume no cooperativity in any of the bindings. <br />
<br />
'''The Equations'''<br />
<br />
[[Image:IC07 Model2.png|thumb|center|600px|'''Model 2''', an energy-dependent network, where the dependence on energy assumes Hill-like dynamics]]<br />
<br clear="all"><br />
<br />
where [E] represents the [nutrient] or ["energy"] within the system. The energy dependence is assumed to follow Hill-like Dynamics. <br />
<br />
<br />
The parameters of our model are described in the table below<br />
<br />
====''Model Parameters''====<br />
<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Parameter <br />
! Description<br />
|-<br />
|<font color = blue>''Kinetic <br> Constants'' </font><br />
|<br />
|-<br />
| k<sub>1</sub><br />
| Maximal constitutive transcription of LuxR by pTET<br />
|-<br />
|k<sub>2</sub><br />
|Binding between LuxR and AHL<br />
|-<br />
|k<sub>3</sub><br />
|Dissociation of protein complex LuxR-AHL (A)<br />
|-<br />
|k<sub>4</sub><br />
|Binding between A and pLux promoter<br />
|-<br />
|k<sub>5</sub><br />
|Dissociaton of A-pLux complex<br />
|-<br />
|k<sub>6</sub><br />
|Transcription of GFP - k<sub>6</sub> proportional to amount of DNA in solution<br />
|-<br />
|<font color = blue>''Degradation <br> Rates'' </font><br />
|<br />
|-<br />
|&delta;<sub>LuxR</sub><br />
|Degradation rate of LuxR<br><br />
&delta;<sub>LuxR</sub> is negligible – assumed to be zero<br />
|-<br />
|&delta;<sub>AHL</sub><br />
|Degradation rate of AHL<br><br />
&delta;<sub>AHL</sub> is negligible – assumed to be zero<br />
|-<br />
|&delta;<sub>GFP</sub><br />
|Degradation rate of GFP<br><br />
&delta;<sub>GFP</sub> is negligible – assumed to be zero<br />
|-<br />
|<font color = blue>''Hill Co-operativity''</font><br />
|<br />
|-<br />
|n<br />
|Co-operativity coefficient describing the degree <br>of energy dependence, which follows Hill-like dynamics<br />
|-<br />
|<font color = blue>''Energy consumption <br> of transcription''</font> <br />
|<br />
|-<br />
|&alpha;<sub>1</sub><br />
|Energy consumption due to constitutive transcription of LuxR<br />
|-<br />
|&alpha;<sub>2</sub><br />
|Energy consumption due to transcription of ''gfp'' gene<br />
|-<br />
|<font color = blue>''Initial Conditions''</font> <br />
|<br />
|-<br />
|P<sub>0</sub><br />
|Initial Concentration of Promoters <br><br />
P<sub>0</sub> represents the amount of DNA inserted into the extract<br />
|-<br />
|LuxR<sub>0</sub><br />
|Initial Concentration of LuxR<br />
<br />
|}<br />
<br />
<br><br />
<br />
We now present the essential features of the system behaviour, simulated for a given set of parameters.<br />
<br />
==Simulations==<br />
<br />
Presented below are the most essential results of the simulations performed. <br />
<br />
In light of the rapid equilibrium approximation which was employed in developing the model, relatively high k-values were selected: <br />
<br>k2 = k3 = k4 = k5 = 100 (dynamical equilibrium); k1 = 0.3 (constitutive transcription by pTet); k6 = 20*k1 (transcription of ''gfp'');<br />
<br />
<br />
<br />
==== 1. Construct 1: [GFP] vs time - varying [AHL]<sub>0</sub>====<br />
<br />
[[Image: C1 AHL GFP.png|thumb|left|430px|'''Figure 5''': Investigation of the effect on GFP expression by <br>'''Construct 1''', when the initial [AHL] is varied i.e. AHL<sub>0</sub> = 0.1, 1, 5, 10, 50 & 100 a.u. (arbitrary units)]]<br />
<br />
[[Image: C1 AHL Energy.png|thumb|right|430px|'''Figure 6''': Comparison of energy consumption of '''Construct''' 1, when the initial [AHL] is varied i.e. AHL<sub>0</sub> = 0.1, 1, 5, 10, 50 & 100 a.u (arbitrary units)]]<br />
<br />
====Discussion====<br />
<br />
Figures 5 and 6 illustrate GFP expression and Energy depletion of construct 1, at various initial AHL concentrations.<br />
<br />
The absolute value of the peak expression is a function of the various rate constants. Here our analysis serves to illustrate the general behaviour offered by construct 1 – a qualitative approach.<br />
<br />
As initial [AHL] is increased, the level of expression increases accordingly to a point were there is negligible difference between the maximal outputs between adjacent tested cases of [AHL]. In fact, from this figure, and for this set of parameters, it is suggestive that saturation occurs at approximately 5 a.u.; in fact, the difference in maximal GFP output between when AHL is increased a several-fold is less than 10%. <br />
<br />
Figure 6, the energy depletion plot, serves to illustrate the effect of increased initial concentrations of AHL on consumption of energy. More resources (promoters) need to be employed to "accommodate" the increasing [AHL]. This obviously increases the rate of energy depletion, until it levels off, as saturating behaviour has been attained - promoter saturation. This is the likely explanation for the "saturation curve" obtained from the experimental data, since the protein degradation terms themselves are almost negligible.<br />
<br />
====2. Construct 1: [GFP] vs [AHL] - transfer function curve====<br />
<br />
{|-<br />
| [[Image: C1 transfer 300min.png|thumb|left|430px|'''Figure 7''': Transfer function of construct 1, after t = 300 min(x-axis is log-scaled). [AHL] measured in arbitrary units]] || [[Image: C1 Transfer 1000min.png|thumb|right|430px|'''Figure 8''': Transfer function of construct 1, after t = 1000min (x-axis is log-scaled) [AHL] measured in arbitrary units]]<br />
|}<br />
<br clear="all"><br />
<br />
====Discussion====<br />
Figure 7 serves to amplify the essential claims from the previous simulation. Saturative behaviour occurs at approximately [AHL] = 5-10 arbitrary units (a.u.). The effect of increasing initial [AHL] beyond this level is of negligible effect on [GFP] expression, for initially available resources. This first transfer function is taken at t=300min. <br />
We would expect the curve to be considerably reduced at a later instance. This is due to reduced energy resources. This ties in with what is observed in figure 8, at t = 1000 above: significantly reduced peak expression of GFP.<br />
<br />
====Simulation 3 – effect of varying GFP degradation term on dynamic behaviour of construct 1====<br />
[[Image: C1 dGFP.png|thumb|left|450px|'''Figure 9''': ]]<br clear="all"><br />
<br />
====Discussion====<br />
This simulation illustrates the effect of delta_GFP on the expression of GFP by construct 1. At small, delta_GFP, the steady-state approach clearly can be attributed to the considerable rate of energy depletion. When the degradation terms are more significant, their effect is considerably greater than the effect of limited resources.<br />
If our model were not energy-dependent, then the simulation for &delta;<sub>GFP</sub> tending to zero should yield positively sloped parabolic curve.<br />
<br />
=== Conclusions ===<br />
<br />
From the initial simulations performed, it was suggested that construct 2 was more effective than construct 1 on the basis of sensitivity, response time and maximal output of reporter. Construct 1 was superior in terms of energy-efficiency. Its life-time is far greater than that of its counterpart. <br />
<br />
Experimental work was performed on both constructs. The notable feature of these endeavours was that Construct 2 was eventually aborted due to difficulty in effectively purifying LuxR. Since our initial simulations are promising with regards to this device, it should be investigated more fully in the future.<br />
In any case, the simulations for construct 1, tie in with that presented by the experimental team, and are a fullfilment of our specifications.<br />
<br />
===Software===<br />
<br />
All deterministic simulations were performed using Matlab 7 (The MathWorks Inc., Natick, MA).<br />
*m-files of all simulations are available on our [https://2007.igem.org/Imperial/Dry_Lab/Software Software] page.<br />
<br />
===References===<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Design << Design] | Modelling | [https://2007.igem.org/Imperial/Infector_Detector/Implementation Implementation >>]<br />
</center></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Cell_by_Date/ConclusionImperial/Cell by Date/Conclusion2007-10-26T23:15:54Z<p>Dirkvs: /* Cell by Date: Conclusion */</p>
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<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 />
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__NOTOC__<br />
= Cell by Date: Conclusion =<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 />
|<center>'''Achieved ?'''</center><br />
|-<br />
|style="background:#ffffcc"|Inputs<br />
|<center>Isothermal conditions between 0 & 40 C</center><br />
||'''<font color=green>4&deg;C shows negligible activity while 37&deg;C shows highest fluorescence <br />
<br />
|-<br />
|style="background:#ffffcc"|<br />
|<center>Dynamic conditions eg. steps & ramps</center><br />
||'''<font color=green>The system responded to stepping of temperature up and down</font>''' <br />
|-<br />
|style="background:#ffffcc"|Outputs<br />
|<center>System should give a visual signal to indicate level of thermal exposure where beef is off</center><br />
|'''<font color=red>Future work- DSred Express can be substituted for GFPmut3b</font>''' <br />
|-<br />
|style="background:#ffffcc"|Activation Energy<br />
|<center>System needs to have an activation Energy 30 +/- 20 kJ/mol</center><br />
||'''<font color=red>Activation energy of system was calculated to be 1.5kJ/mol</font>''' <br />
|-<br />
|style="background:#ffffcc"|Health Regulations<br />
|<center>System must not be living replicating bacteria</center><br />
|<center>'''<font color=green>Yes through use of cell free chassis</font>''' </center><br />
|-<br />
|style="background:#ffffcc"|Response Time<br />
|<center>System needs to have a response time under 1 hour</center><br />
||'''<font color=green>Yes system increases significantly after 1 hour</font>''' <br />
|-<br />
|style="background:#ffffcc"|Lifespan<br />
|<center>System must have a shelf life of 7 days</center><br />
||'''<font color=red>Not Determined- Packaging method that prevents evaporation</font>''' <br />
|}<br />
<br clear="all"><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 />
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<center> [https://2007.igem.org/Imperial/Cell_by_Date/Testing << Testing ] | Conclusions | [https://2007.igem.org/Imperial Home >> ]</center></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Cell_by_Date/SpecificationImperial/Cell by Date/Specification2007-10-26T23:13:12Z<p>Dirkvs: /* Cell by Date: Specifications */</p>
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<ul><br />
<li><a href="https://2007.igem.org/Imperial/Cell_by_Date/Introduction" title=""><span>Introduction</span></a></li><br />
<li><a class="current" 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 href="https://2007.igem.org/Imperial/Cell_by_Date/Conclusion" title=""><span>Conclusion</span></a></li><br />
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__NOTOC__<br />
= Cell by Date: Specifications =<br />
<br />
'''Cell by Date in the Cold Chain:'''<br />
<br />
<br />
[[Image:CBDCartoon.png]]<br />
<br />
<br clear="all"><br />
<br />
{|<br />
|-valign="top"<br />
|width=10%|[[Image:CBDthermometer.gif|center]]<br><br><br><br><br><br><br><br>[[Image:CBDCartoonHamburger1.png|center]]<br clear ="all"><br><br><br><br><br><br>[[Image:CBDQuestionMarkCartoon.png|center]]<br><br />
|width=30%|[[Image:CBDSpecs.png]]<br />
|width=60%|<br />
*'''Input: Isothermal & Dynamic conditions, eg. steps & ramps between 0-40 &deg;C'''<br />
Temperature is considered to be the major factor in beef spoilage and although the meat industry tries to keep temperature low during transportaion temperature conditions higher than 10&deg;C are not unusual during transport<sup>[[#References |3]]</sup>. It is therefore important that our that we look at the performance of our system in isothermal conditions eg. in the cold chain, and dynamic temperature scenarios eg. a break in the cold chain.<br />
*'''Output: Visible Signal'''<br />
Several Technologies already exist which address the problem of monitoring the thermal exposure of products in the cold chain. One particular family are Temperature Time Integrators (TTIs).<sup>[[#References |5]]</sup><br><br />
The key aspect of a TTI is that they are based on a phenomenon which can act as a signal to a consumer for example, eg. a colour change. For Cell By Date we would like a visible signal that tells the consumer that as a result of the thermal history of the beef it is not fit for consumption e.g. it's been left out of the fridge for too long. To Determine the point at which ground beef is not good to eat we have to look at the biology of the spoilage process.The dominant organisms leading to the spoilage of beef depend of the beef's composition and the environmental conditions under which the beef is stored. For refrigerated packaged beef Pseudomonas spp. were dominate areobically while Lactobacillus was dominant anaerobically.<sup>[[#References |1]]</sup> <br />
<br />
*'''Lifespan : 7 days'''<br />
There also seems to be a general rule for beef that when the bacterial count reaches 10<sup>7</sup> cm<sup>-2</sup>, off odours and slime production occur and the beef is considered off. <sup>[[#References |2]]</sup> For controlled isothermal conditions in a laboratory environment the time taken for beef to reach this spoilage point seems to be at most 7 days.<sup>[[#References |3]]</sup> This implies that the shelf life of our system needs to be at least 7 days.<br />
<br />
*'''Health & Safety : Non-living , non-infectious'''<br><br />
Because bacteria are responsible for the spoilage of beef it is unwise to use a bacteria based device eg. by using e.coli/yeast as a chassis as this would further add to the spoilage.<br />
<br />
*'''Activation Energy : 30 +/- 20 kJ/mol'''<br><br />
<br />
The Gompertz's model is widely used when considering beef spoilage as it has been shown to fit growth data very well. <sup>[[#References |1]]</sup> Using the Gompertz model we can get the specific growth rate, Lag phase duration (LPD) and maximum population density (MPD) for bacterial growth at a particular constant temperature. And then using these we can determine the Activation Energy (Ea) for the beef spoilage reaction. For U2 grade Argentinian beef stored in polyethylene and SARAN PVC, the Ea ranged from 80kJ mol<sup>-1</sup> to 220kJ mol<sup>-1</sup> for a range of bacteria. <sup>[[#References |4]]</sup><br />
<br />
One contrary value for the Ea of the beef spoilage reactions is given by Leak <sup>[[#References |2]]</sup> who calculated Ea = 30kJ mol<sup>-1</sup>. The difference between Leak's value and that of Giannuzzi <sup>[[#References |4]]</sup> probably lies in their packaging methods, which is of importance as the project aims to target aerobically fresh gound beef.<br><br />
<br />
In order for Cell by Date to act as a TTI & accurately report the spoilage rxn of beef, the activation energy of the two rxns needs to be similar. For example a difference between the two Ea's less than 20kJ mol<sup>-1</sup> would result in the TTI estimating the thermal history of the beef to be within 1&deg;C of the actual history.<sup>[[#References |6]]</sup> For Cell by Date we would like our Activation Energy of our system to be 30 +/- 20 kJ mol<sup>-1</sup> to yield an accurate thermal history of the most sensitive spoilage rxn found by Leak, which is most likely for aerobically fresh beef.<br />
<br />
*'''Response Time: Less that 1 Hour'''<br><br />
In addition, to correctly couple the Ea of our system to that of the dominant spoilage reaction in beef, we also have to consider the response time of our sytem. Our system needs to have a rapid response time, so in other words it needs to be able to quickly switch between states eg. low output and high output. This is so our system can capture small variations of temperature in the cold chain and report them in a meangingful way. Specifically, a response time in the order of a few hours would ensure that if there are any problems in the cold chain this will arise in our system very quickly.<br />
|}<br />
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<br />
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<center> [https://2007.igem.org/Imperial/Cell_by_Date/Introduction << Introduction ] | Specifications | [https://2007.igem.org/Imperial/Cell_by_Date/Design Design >>]</center><br />
<br />
<br />
== References ==<br />
# [http://www.springerlink.com/index/G311226U2M51T844.pdf Labuza TP, Fu B. Growth kinetics for shelf-life prediction: theory and practice. J of Industrial Microbiology 1993;12:309-23.]<br />
# [http://66.102.1.104/scholar?hl=en&lr=&q=cache:thi6BTIW1YMJ:www.vitsab.com/PDF/V507.pdf+TTI+Beef+Activation+Energy Leak, F.W. (2000): Quality changes in Ground beef during distribution and storage, and determination of Time- Temperature-Indicator (TTI) charakteristic of ground beef University of Florida Institute of food and Agricultural Sciences Internet: www.vitsab.com, Stand: April 2003]<br />
# [http://iufost.edpsciences.org/index.php?option=article&access=standard&Itemid=129&url=/articles/iufost/pdf/2006/01/iufost06000765.pdf Koutsoumanis K, Stamatiou A, Skandamis P, Nychas GJ. Development of a Microbial Model for the Combined Effect of Temperature and pH on Spoilage of Ground Meat, and Validation of the Model under Dynamic Temperature Conditions. Appl Environ Microbiol. 2006 Jan;72(1):124-34.]<br />
# [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T7K-3S3M25D-B&_user=217827&_coverDate=01%2F06%2F1998&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000011279&_version=1&_urlVersion=0&_userid=217827&md5=80630ba21fbc6869b9f5179d334734da Giannuzzi L, Pinotti A, Zaritzky N. Mathematical modelling of microbial growth in packaged refrigerated beef stored at different temperatures. Int J Food Microbiol. 1998 Jan 6;39(1-2):101-10.]<br />
# [http://www.iaph.uni-bonn.de/Coldchain/downloads/06_Labuza.pdf Labuza 2006 : Cold Chain-Management II Time-temperature Integrators and the Cold chain: What is next? Bonn Germany 5/8/06]<br><br />
# [http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1365-2621.2001.tb15211.x?cookieSet=1 E. Shimoni, E.M. Anderson, T.P. Labuza (2001) Reliability of Time Temperature Indicators Under Temperature Abuse. Journal of Food Science 66 (9), 1337–1340. doi:10.1111/j.1365-2621.2001.tb15211.x ]<br />
# [http://linkinghub.elsevier.com/retrieve/pii/S0168-1605(05)00067-X Giannakourou MC, Koutsoumanis K, Nychas GJ, Taoukis PS. Field evaluation of the application of time temperature integrators for monitoring fish quality in the chill chain. Int J Food Microbiol. 2005 Jul 25;102(3):323-36.]</div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Cell_by_Date/IntroductionImperial/Cell by Date/Introduction2007-10-26T23:12:19Z<p>Dirkvs: /* Cell by Date in the Cold Chain */</p>
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<ul><br />
<li><a class="current" 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 href="https://2007.igem.org/Imperial/Cell_by_Date/Conclusion" title=""><span>Conclusion</span></a></li><br />
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__NOTOC__<br />
<br />
<br />
==Cell by Date Summary ==<br />
Cell by Date is a Temperature Time Integrator, and will report when ground beef has been out of the cold chain for too long (the cold chain being the temperature controlled transport chain from abbatoire to supermarket). Cell By Date does this by exploiting the thermal dependence of gene expression systems to express fluorescent proteins at ambient temperatures above 8°C. By using simple constructs, Cell by Date allows the characterisation of the various cell-free chassis, enhancing our understanding of this foundational technology. <br />
<br />
====Cell by Date in the Cold Chain====<br />
<br />
[[Image:CBDCartoon.png]]<br />
<br />
== Motivations ==<br />
<br />
The hamburger has become synonymous with modern western culture. We see it in fast food chains , ready-made packages in supermarkets, and at most home barbeques - all made using ground beef. But who controls the quality of our burgers, who ensures that the burgers we eat don’t spoil the fun and make us sick? <br />
<br />
===== Ensuring the Freshness of Beef =====<br />
A lot of work has been carried out to study the spoilage of the beef that make up our burgers. In fact, there are professional organisations such as FDA and HACCAP that give strict regulations to ensure the quality of beef that meat industries produce so that we don't end up with burgers like the ones below ! <br />
<br />
[[Image:IC07_burger.png|center|600px]]<br />
<br><br />
<br />
As consumers, we can see this in our supermarkets in the form of a printed ‘sell by date’, which is based upon challenge testing - a process in which beef is put through numerous temperature scenarios and a prediction then made about how long the beef will last after it has left the factory. Due to safety reservations, this prediction made on printed sell by date often indicates that the beef is off when in may in fact have a few days left. <br />
<br />
On the other hand, survey studies have shown that temperature conditions higher than 10°C are not unusual during transportation, retail storage, and consumer handling of beef products. Such temperature abuses during any stage of the cold chain could result in an unexpected loss of quality and a significant decrease of shelf life. Indeed, printed sell by date labels are seldom the best estimate of meat shelf-life. To combat this, the meat industry has looked into other ways of spoilage prediction and managing the cold chain.<br />
<br />
===== Temperature Time Integrators =====<br />
[[Image:TTIs.gif|frame|300px|right|commercially available TTIs]]<br />
One particular tool used to monitor the cold chain is a family of devices called temperature time integrators (TTIs). These devices try to monitor the thermal exposure of the food. When a certain level of exposure is reached, a change occurs, usually visual, which signals that the beef has gone off.<br />
<br />
This project is concerned with the development of a TTI that is based upon synthetic biology components; exploiting the thermal dependence of gene expression of simple reporter system in a cell free extract.<br />
<br />
With an idea of the problem we are trying to target head on to our specification page where we take the first step in our engineering cycle to ensure the quality, relevance and robustness of our solution.<br />
<br><br><br><br><br><br />
<br />
== Achievements ==<br />
* We designed and modelled a device to indicate spoilage of meat/break in the cold chain via the thermal history of the system.<br />
* We successfully characterised pTet GFP and pT7 GFP under isothermal conditions.<br />
* We further characterised pTet GFP under dynamic temperature conditions.<br />
* The results of the Cell by Date project were to characterise cell-free chassis using a resource-dependent model.<br />
<br />
<br />
<br />
<center> On to next stage : | [https://2007.igem.org/Imperial/Cell_by_Date/Specification Specifications >>]<br />
</center><br />
<br />
== References ==<br />
<br />
Food Hygiene, Microbiology and HACCP <br><br />
[http://www.sensitech.nl/en/index.html Sensitech Company]<br><br />
[http://www.sensitech.nl/en/coldchain_info/index.html Sensitech Cold Chain Background]<br><br />
[http://www.sensitech.nl/en/products/index.html Sensitech Products]<br><br />
[http://www.globalcoldchain.com/ Global Cold Chain Company]<br><br />
[http://www.who.int/vaccines-documents/DocsPDF/www9804.pdf WHO : Temperature Monitors for vaccines and the Cold Chain]<br><br />
[http://www.foodproductiondaily.com/news/ng.asp?id=51051-diagnostic-packaging-potential FoodProductionDaily.com Overview of Industry]<br><br />
[http://www.packaging-gateway.com/downloads/conferences/PACE/PackagingBrody_Inc_Prsntation_4.pdf Brody 2006 Good overview of industry including active packaging]<br><br />
[http://www.foodtechsource.com/emag/003/trend.htm Interview with Labuza about food law]<br><br />
[http://aem.asm.org/cgi/reprint/72/7/4663 Ercolini,2006 : 5 Degrees Isothermal conditions varying Packaging]</div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/ProtocolsImperial/Wet Lab/Protocols2007-10-26T23:08:41Z<p>Dirkvs: /* Wet Lab: Protocols */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
__NOTOC__<br />
<br />
= Wet Lab: Protocols =<br />
Below is a databank of all the protocols that have been used in our projects. <br />
<br><br><br />
<br />
{| cellpadding="5" style="background:#F5FAFF; border: 1px solid #aabadd; color:Black" align="left"<br />
|- style="background:#aabadd; color:Black" align="center" <br />
||<font size=2>'''General Experiments'''</font>||<font size=2>'''Testing Experiments'''</font><br />
|- style="background:#d5dcef; color:Black" valign="top"<br />
|<br />
<font size=2><font color=green>'''Protein Purification'''</font> <br><br />
* ''Purification of LuxR'': [[Imperial/Wet_Lab/Protocols/Prot1.5|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.5|Results]] <br />
* ''Purification of GFPmut3b'': [[Imperial/Wet_Lab/Protocols/Prot1.6|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.6|Results]]<br />
<br><br />
<font size=2><font color=green>'''Fluorometer Testing'''</font> <br><br />
* ''Optimisation of Fluorometer'': [[Imperial/Wet_Lab/Protocols/Prot1.1|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.1|Results]]<br><br />
<font size=2><font color=green>'''Calibration Curve'''</font> <br><br />
* ''Calibration Curve for GFPmut3b'': [[Imperial/Wet_Lab/Protocols/Prot1.3|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.3|Results]]<br />
* ''Degradation Curve for GFPmut3b'':[[Imperial/Wet_Lab/Protocols/Prot1.4|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.4|Results]]<br />
<br><br />
<font size=2><font color=green>'''Chassis Protocols'''</font> <br><br />
* ''Reaction Mixture of Commercially available S30 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.3|Protocol]]<br />
* ''Reaction Mixture of Homemade S30 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.4|Protocol]]<br />
* ''Recipe for S30 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.1|Protocol]]<br />
* ''Recipe for S12 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.2|Protocol]]<br />
* ''Testing of Homemade S30 Cell Extract with ID construct '':[[Imperial/Wet_Lab/Protocols/CE1.5|Protocol]] and [[Imperial/Wet_Lab/Results/Chas1.1|Results]]<br><br />
* ''Vesicles Formation'': [[Imperial/Wet_Lab/Protocols/VF1.1|Protocol]]<br />
<br><br />
|<br />
<font size=2><font color=green>'''pT7 Characterisation'''</font> <br><br />
*''pT7 in vitro:'' [[Imperial/Wet_Lab/Protocols/Prot1.9|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.9|Results]]<br />
<br><br />
<br />
<font size=2>'''Cell by Date'''</font> <br><br />
<font color=green>'''Implementation'''</font><br><br />
* ''In Vivo Testing:'' [[Imperial/Wet_Lab/Protocols/CBD1.1|Protocols]] and [[Imperial/Wet_Lab/Results/CBD1.1|Results]]<br />
* ''Initial In vitro Testing'': [[Imperial/Wet_Lab/Protocols/CBD1.2|Protocols]] and [[Imperial/Wet_Lab/Results/CBD1.2|Results]]<br />
<font color=green>'''Testing/Validation<br>'''</font><br />
*''Testing DNA Concentration in vitro'': [[Imperial/Wet_Lab/Protocols/CBD1.3|Protocols]] and [[Imperial/Wet_Lab/Results/Res1.8|Results]]<br />
* ''Isothermal experiments'': [[Imperial/Wet_Lab/Protocols/CBD2.2|Protocols]] and [[Imperial/Wet_Lab/Results/CBD2.2|Results]]<br />
*''Temperature Steps:'' [[Imperial/Wet_Lab/Protocols/CBD2.3|Protocols]] and [[Imperial/Wet_Lab/Results/CBD2.3|Results]] <br />
*''Packaging experments:'' [[Imperial/Wet_Lab/Protocols/CBD2.1|Protocols]] and [[Imperial/Wet_Lab/Results/CBD2.1|Results]]<br />
<br><br />
<br />
<font size=2>'''Infector Detector'''</font> <br><br />
<font color=green>'''Implementation<br>'''</font><br />
*''In vivo Testing'': [[Imperial/Wet_Lab/Protocols/ID1.1|Protocol]] and [[Imperial/Wet_Lab/Results/ID1.1|Results]]<br />
* ''Initial In Vitro Testing'': [[Imperial/Wet_Lab/Protocols/ID1.2|Protocol]] and [[Imperial/Wet_Lab/Results/ID1.2|Results]]<br />
<font color=green>'''Testing/Validation<br>'''</font><br />
*''Testing of DNA concentration'' : [[Imperial/Wet_Lab/Protocols/ID2.1|Protocol]] and [[Imperial/Wet_Lab/Results/ID2.1|Results]]<br />
* ''Testing of Purified LuxR'': [[Imperial/Wet_Lab/Protocols/Prot1.7|Protocol]] and [[Imperial/Wet_Lab/Results/ID1.7|Results]]<br />
* ''Characterization in vitro - AHL titration '': [[Imperial/Wet_Lab/Protocols/ID3.1|Protocol]] and [[Imperial/Wet_Lab/Results/ID3.1|Results]]<br />
|}<br />
<br clear="all"></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/ProtocolsImperial/Wet Lab/Protocols2007-10-26T23:05:49Z<p>Dirkvs: /* Wet Lab: Protocols */</p>
<hr />
<div>{{Template:IC07navmenu}}<br />
__NOTOC__<br />
<br />
= Wet Lab: Protocols =<br />
Below is a databank of all the protocols that have been used in our projects. <br />
<br><br><br />
<br />
{| cellpadding="5" style="background:#F5FAFF; border: 1px solid #aabadd; color:Black" align="left"<br />
|- style="background:#aabadd; color:Black" align="center" <br />
||<font size=2>'''General Experiments'''</font>||<font size=2>'''Testing Experiments'''</font><br />
|- style="background:#d5dcef; color:Black" valign="top"<br />
|<br />
<font size=2><font color=green>'''Protein Purification'''</font> <br><br />
* ''Purification of LuxR'': [[Imperial/Wet_Lab/Protocols/Prot1.5|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.5|Results]] <br />
* ''Purification of GFPmut3b'': [[Imperial/Wet_Lab/Protocols/Prot1.6|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.6|Results]]<br />
<br><br />
<font size=2><font color=green>'''Fluorometer Testing'''</font> <br><br />
* ''Optimisation of Fluorometer'': [[Imperial/Wet_Lab/Protocols/Prot1.1|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.1|Results]]<br><br />
<font size=2><font color=green>'''Calibration Curve'''</font> <br><br />
* ''Calibration Curve for GFPmut3b'': [[Imperial/Wet_Lab/Protocols/Prot1.3|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.3|Results]]<br />
* ''Degradation Curve for GFPmut3b'':[[Imperial/Wet_Lab/Protocols/Prot1.4|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.4|Results]]<br />
<br><br />
<font size=2><font color=green>'''Chassis Protocols'''</font> <br><br />
* ''Reaction Mixture of Commercially available S30 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.3|Protocol]]<br />
* ''Reaction Mixture of Homemade S30 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.4|Protocol]]<br />
* ''Recipe for S30 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.1|Protocol]]<br />
* ''Recipe for S12 Cell Extract '':[[Imperial/Wet_Lab/Protocols/CE1.2|Protocol]]<br />
* ''Testing of Homemade S30 Cell Extract with ID construct '':[[Imperial/Wet_Lab/Protocols/CE1.5|Protocol]] and [[Imperial/Wet_Lab/Results/Chas1.1|Results]]<br><br />
* ''Vesicles Formation'':[[Imperial/Wet_Lab/Protocols/VF1.1|Protocol]]<br />
<br><br />
|<br />
<font size=2><font color=green>'''pT7 Characterisation'''</font> <br><br />
*''pT7 in vitro:'' [[Imperial/Wet_Lab/Protocols/Prot1.9|Protocol]] and [[Imperial/Wet_Lab/Results/Res1.9|Results]]<br />
<br><br />
<br />
<font size=2>'''Cell by Date'''</font> <br><br />
<font color=green>'''Implementation'''</font><br><br />
* ''In Vivo Testing:'' [[Imperial/Wet_Lab/Protocols/CBD1.1|Protocols]] and [[Imperial/Wet_Lab/Results/CBD1.1|Results]]<br />
* ''Initial In vitro Testing'': [[Imperial/Wet_Lab/Protocols/CBD1.2|Protocols]] and [[Imperial/Wet_Lab/Results/CBD1.2|Results]]<br />
<font color=green>'''Testing/Validation<br>'''</font><br />
*''Testing DNA Concentration in vitro'': [[Imperial/Wet_Lab/Protocols/CBD1.3|Protocols]] and [[Imperial/Wet_Lab/Results/Res1.8|Results]]<br />
* ''Isothermal experiments'': [[Imperial/Wet_Lab/Protocols/CBD2.2|Protocols]] and [[Imperial/Wet_Lab/Results/CBD2.2|Results]]<br />
*''Temperature Steps:'' [[Imperial/Wet_Lab/Protocols/CBD2.3|Protocols]] and [[Imperial/Wet_Lab/Results/CBD2.3|Results]] <br />
*''Packaging experments:'' [[Imperial/Wet_Lab/Protocols/CBD2.1|Protocols]] and [[Imperial/Wet_Lab/Results/CBD2.1|Results]]<br />
<br><br />
<br />
<font size=2>'''Infector Detector'''</font> <br><br />
<font color=green>'''Implementation<br>'''</font><br />
*''In vivo Testing'': [[Imperial/Wet_Lab/Protocols/ID1.1|Protocol]] and [[Imperial/Wet_Lab/Results/ID1.1|Results]]<br />
* ''Initial In Vitro Testing'': [[Imperial/Wet_Lab/Protocols/ID1.2|Protocol]] and [[Imperial/Wet_Lab/Results/ID1.2|Results]]<br />
<font color=green>'''Testing/Validation<br>'''</font><br />
*''Testing of DNA concentration'' : [[Imperial/Wet_Lab/Protocols/ID2.1|Protocol]] and [[Imperial/Wet_Lab/Results/ID2.1|Results]]<br />
* ''Testing of Purified LuxR'': [[Imperial/Wet_Lab/Protocols/Prot1.7|Protocol]] and [[Imperial/Wet_Lab/Results/ID1.7|Results]]<br />
* ''Characterization in vitro - AHL titration '': [[Imperial/Wet_Lab/Protocols/ID3.1|Protocol]] and [[Imperial/Wet_Lab/Results/ID3.1|Results]]<br />
|}<br />
<br clear="all"></div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-09-04Imperial/Wet Lab/Lab Notebook/2007-09-042007-10-26T22:57:43Z<p>Dirkvs: /* GFP Calibration Curve */</p>
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<div>{{Template:IC07navmenu}}<br />
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__NOTOC__<br />
= 4 September 2007 =<br />
<br />
<br />
==Construction of pT7-GFP==<br />
#Picked colonies to grow in 5ml LB + Kan<br />
#Cultures left to grow at 37&deg;C overnight<br />
==GFP Calibration Curve==<br />
More concentrations were carried out for the calibration curve, these give a range of concentrations tested:<br />
*3.7uM, 1.8uM, 1.uM, 0.5uM, 0.1uM, 0.05uM, 0.025uM, 0.0125uM <br />
The calibration curve can be found [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Experimental_Design/Phase2/Results_1.2 Here]<br />
<br />
== Vesicles ==<br />
'''Formation of Vesicles'''<br />
<br />
2160&mu;l of Solution A-CE was prepared with the following components:<br />
* 583&mu;l of S30 cell extract (home made)<br />
* 1080&mu;l of cell extract buffer<br />
* 36&mu;l of rNTP solution<br />
* 112&mu;l of Pyruvate Kinase solution<br />
* 349&mu;l of ddH<sub>2</sub>O<br />
<br />
<br />
360&mu;l of Solution B-CE was prepared with the following components:<br />
* 351&mu;l of Solution A-CE<br />
* 4&mu;l of GFP solution<br />
* 5&mu;l of DNA solution (P<sub>tet</sub> with GFP)<br />
<br />
The remaining 1.8ml of Solution A-CE was then diluted 10x to form 18ml of Solution A-CEd<br />
<br />
The following 3 emulsions were prepared:<br />
*50&mu;l of Solution B-CE into 8ml of POPC/dodecane suspension<br />
*50&mu;l of Solution B-CE into 8ml of DOPC/dodecane suspension<br />
*250&mu;l of Solution B-CE into 47,8ml of POPC/dodecane suspension<br />
<br />
<br />
11 samples were created in total (note there is no Sample 9):<br />
<br />
* '''Sample Noireaux''': Produced from the [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.3 | Noireaux protocol]. Solution B-CE was added to 200&mu;l taken from the 50ml POPC/dodecane suspension prepared [[Imperial/Wet Lab/Lab Notebook/2007-09-03|the day before]].<br />
* '''Sample 1''': 2ml from the 10ml POPC/dodecane suspension was used to prepare the interface over 3ml of Solution A-CEd. 1ml of emulsion was added 2 hours later, centrifuged at 120x g, and collected.<br />
* '''Sample 2''': 2ml from the 10ml DOPC/dodecane suspension was used to prepare the interface over 3ml of Solution A-CEd. 1ml of emulsion was added 2 hours later, centrifuged at 120x g, and collected.<br />
* '''Sample 3''': Prepared [[Imperial/Wet Lab/Lab Notebook/2007-09-03|the day before]] and left overnight before being collected. Sample not centrifuged.<br />
* '''Sample 4''': Prepared [[Imperial/Wet Lab/Lab Notebook/2007-09-03|the day before]] and left overnight before being collected. Sample not centrifuged.<br />
* '''Sample 5''': 2ml from the 50ml POPC/dodecane suspension was used to prepare the interface over 3ml of Solution A-CEd. 1ml of emulsion was added 2 hours later, centrifuged at 120x g, and collected.<br />
* '''Sample 6''': 2ml from the 10ml POPC/dodecane emulsion was poured over 3ml of Solution A-CEd (with no prepared interface), and left to rest for 2 hours, before being centrifuged at 120x g for 10 minutes and collected.<br />
* '''Sample 7''': 2ml from the 10ml DOPC/dodecane emulsion was poured over 3ml of Solution A-CEd (with no prepared interface), and left to rest for 2 hours, before being centrifuged at 120x g for 10 minutes and collected.<br />
* '''Sample 8''': 2ml from the 50ml POPC/dodecane emulsion was poured over 3ml of Solution A-CEd (with no prepared interface), and left to rest for 2 hours, before being centrifuged at 120x g for 10 minutes and collected.<br />
* '''Sample 10''': 2ml from the 10ml POPC/dodecane emulsion recycled from [[Imperial/Wet Lab/Lab Notebook/2007-09-03|the day before]] was left stirring overnight, and then was poured over 3ml of Solution A (with no prepared interface). It was left to rest for 2 hours before being centrifuged at 120x g for 10 minutes and collected.<br />
* '''Sample 11''': 2ml from the 10ml DOPC/dodecane emulsion recycled from [[Imperial/Wet Lab/Lab Notebook/2007-09-03|the day before]] was left stirring overnight, and then was poured over 3ml of Solution A (with no prepared interface). It was left to rest for 2 hours before being centrifuged at 120x g for 10 minutes and collected.<br />
<br />
Samples 1-11 were produced using [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.2 | protocol 1.2]<br />
<br />
Samples 1, 2, 5, 6, 7, 8 were prepared from an interface formed on top of 3ml of Solution A-CEd (see above).<br />
<br />
<br />
<br />
<br />
'''Results''' <br><br />
<br />
All 11 samples prepared today were scrutinised under the microscope. Here are the results (scroll down to the bottom for images.):<br />
<br />
* '''Sample Noireaux''': No vesicles were found. There were ''lots'' of GFP aggregates.<br />
* '''Sample 1''': Plenty of vesicles were found, but with very weak fluorescence inside. There were ''lots'' of GFP aggregates.<br />
* '''Sample 2''': No vesicles were found, but one large fluorescent blob was present. There were ''lots'' of GFP aggregates. A strange, unidentified texture of objects was found.<br />
* '''Sample 3''': One possible vesicle or blob found, with no fluorescence inside. E.coli contamination. Strange vesicle coalescence around a large GFP aggregate (vesicles faintly fluorescent).<br />
* '''Sample 4''': Lots of coalescence, with faint fluorescence inside.<br />
* '''Sample 5''': Flocculated vesicles, with very faint fluorescence inside.<br />
* '''Sample 6''': Well formed vesicles, and some coalescence, with faint and not-so-faint fluorescence inside.<br />
* '''Sample 7''': Rare but well formed vesicles, with faint fluorescence inside.<br />
* '''Sample 8''': Best results. High count of well formed vesicles, with not-so-faint fluorescence inside.<br />
* '''Sample 10''': Single large coalesced blob, with faint fluorescence, in spite of dense aggregate. No external aggregates.<br />
* '''Sample 11''': Many 2&mu;m fluorescent vesicles, with very good enclosure - very few external aggregates. Large group of coalescence blobs.<br />
<br />
<br />
<br />
<br />
'''Preparations''' <br><br />
No preparations were made today.<br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image145.jpg|200px]]<br />
| width="200px"| [[image:IC07_image144.jpg|200px]]<br />
| width="200px"| [[image:IC07_image144b.jpg|200px]]<br />
|- <br />
|colspan="3" width="600px"|An example of GFP aggregates, found in Sample Noireaux. Left: Many large GFP aggregates are visible with white light. Centre: The same picture, but with fluorescence. The fluorescence is too weak to be captured by the camera. Right: The same picture as the centre, but with full saturation and gamma correction. The faint fluorescence is picked up by the image enhancement.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image146.jpg|200px]]<br />
| width="200px"| [[image:IC07_image148.jpg|200px]]<br />
| width="200px"| [[image:IC07_image150.jpg|200px]]<br />
|- <br />
|colspan="3" width="600px"|Faint vesicles found in Sample 1. Above: Three different vesicles found in Sample 1, in white light. Below left: The 'above centre' image, with fluorescence switched on. The camera is not able to capture the fluorescence. Below centre and right: The 'above right' image, with fluorescence switched on (centre), and with full saturation and gamma correction (right). There was no fluorescence captured by the camera inside the vesicle, but the large aggregate near the top is visible in the enhanced image.<br />
|-<br />
| width="200px"| [[image:IC07_image147.jpg|200px]]<br />
| width="200px"| [[image:IC07_image149.jpg|200px]]<br />
| width="200px"| [[image:IC07_image149b.jpg|200px]]<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image152.jpg|200px]]<br />
| width="200px"| [[image:IC07_image151.jpg|200px]]<br />
| width="200px"| [[image:IC07_image151b.jpg|200px]]<br />
|- <br />
|colspan="3" width="600px"|The large fluorescent object found in Sample 2. The object had an internal refractive index different from what is usually observed in vesicles or blobs. Left: The object under white light. Centre: The same picture, but with fluorescence. The fluorescence is too weak to be captured by the camera. Right: The same picture as the centre, but with full saturation and gamma correction. The faint fluorescence is picked up by the image enhancement.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image153.jpg|200px]]<br />
|- <br />
|colspan="1" width="200px"|A strange texture found in Sample 2. It was not clear whether these 'dots' were in aqueous solution or a large air bubble under the slide cover.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image154.jpg|200px]]<br />
| width="200px"| [[image:IC07_image155.jpg|200px]]<br />
|- <br />
|colspan="2" width="400px"|Pictures from Sample 3. Left: A large coalescence of vesicles, faintly fluorescent. Right: E.coli contamination from an unidentified source.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image158.jpg|200px]]<br />
| width="200px"| [[image:IC07_image157.jpg|200px]]<br />
| width="200px"| [[image:IC07_image157b.jpg|200px]]<br />
|- <br />
|colspan="3" width="600px"|A large GFP aggregate surrounded by coalescing faintly fluorescent vesicles, found in Sample 3. Left: The object under white light. Centre: The same picture, but with fluorescence. The fluorescence is too weak to be captured by the camera. Right: The same picture as the centre, but with full saturation and gamma correction. The faint fluorescence is picked up by the image enhancement.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image159.jpg|200px]]<br />
|- <br />
|colspan="1" width="200px"|An example of a coalescence blob, found in Sample 4. Note that the refractive index is the same both inside and outside the blob. There also seems to be a presence of small GFP aggregates inside. The object was faintly fluorescent.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image161.jpg|200px]]<br />
|- <br />
|colspan="1" width="200px"|An example of flocculated vesicles, found in Sample 5. The vesicles were faintly fluorescent.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image163.jpg|200px]]<br />
| width="200px"| [[image:IC07_image166.jpg|200px]]<br />
|- <br />
|colspan="2" width="400px"|Faintly fluorescent vesicles found in Sample 6. The fluorescence was too weak to be captured by the camera.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image167.jpg|200px]]<br />
|- <br />
|colspan="1" width="200px"|One of the few, faintly fluorescent, well formed vesicles found in Sample 7. The fluorescence was too weak to be captured by the camera. Note the difference in refractive index between inside and outside the vesicle. The solution inside the vesicle is much more concentrated than outside.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image169.jpg|200px]]<br />
|- <br />
|colspan="1" width="200px"|One of the many fluorescent vesicles found in Sample 8. The fluorescence was too weak to be captured by the camera.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image171.jpg|200px]]<br />
|- <br />
|colspan="1" width="200px"|The single large coalescence blob, with faint fluorescence (in spite of dense aggregate) found in Sample 10.<br />
|}<br clear="all"><br />
<br />
{|align="left" border="1"<br />
| width="200px"| [[image:IC07_image172.jpg|200px]]<br />
| width="200px"| [[image:IC07_image173.jpg|200px]]<br />
|- <br />
|colspan="2" width="400px"| Images from Sample 11. Left: Vesicles and coalescence blobs under white light. Right: Single vesicle, under white light. Both enclosures were fluorescent - though not enough to be caught by the camera.<br />
|}<br clear="all"><br />
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{{Template:IC07labnotebook}}</div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Wet_Lab/Lab_Notebook/2007-08-17Imperial/Wet Lab/Lab Notebook/2007-08-172007-10-26T22:54:11Z<p>Dirkvs: /* Pilot Preparation of Vesicles */</p>
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<div>__NOTOC__<br />
{{Template:IC07navmenu}}<br />
<html><div id="maincol"></html><br />
<br />
= 17 August 2007 =<br />
<br><br><br><br />
==Restriction Digest of Biobricks==<br />
#9 DNA samples were digested by EcoRI and PstI<br />
#4 μl of DNA was digested by 1 μl of enzymes at 37&deg;C for 1 hour<br />
<br />
<br clear="all"><br><br><br><br><br />
==Agarose Gel Electrophoresis of Biobricks==<br />
#Checked 9 Biobricks and 9 Digests (lebelled R) on 1% agarose gel<br />
<br />
Protocols can be found at [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#S30.2FS12_Cell_Extract#Electrophoresis Electrophoresis] in the general protocols page<br />
<br />
[[Image:IC07 Gel 17 8.jpg]]<br clear="all"><br />
Conclusions:<br />
*The brick for cI promoter (sample 14) is likely to be faulty<br />
*There might be a problem with one of the restriction sites of T7-GFP (sample 11)<br />
<br />
==Retesting of pT7 in vivo==<br />
Today we restested the pT7 in vivo, ensureing that we properly induced it. The results were that there was no expression of GFP from pT7. From these results we will carry out further investigation<br />
<br />
==Testing of DNA Constructs in vitro==<br />
<br />
*The following constructs were tested:<br />
#pT7 in Vivo (200 microLitres in each well) -3 repeats<br />
#pTet in Vitro (Homemade cell extract: 100 microL of cell extract + 10 microL of DNA) - 2 repeats<br />
#pT7 in Vitro (Homemade cell extract: 100 microL of cell extract + 10 microL of DNA) - 2 repeats <br />
<br />
*Only 2 repeats were carried out to conserve homemade cell extract<br />
*The experiment was carried out at room temp of 25 <sup>o</sup>C average<br />
*Measurements were taken every 15 min for 4 h<br />
*The fluorometer counting time of the detector was 0.15 s<br />
<br />
==Pilot Preparation of Vesicles==<br />
<br />
*'''Formation of Vesicles''': Two of the four suspensions prepared the [[Imperial/Wet Lab/Lab Notebook/2007-08-16#Pilot Preparation of Vesicles|day before]] (one well-desiccated and one poorly-desiccated) were used to form vesicles:<br />
#2ml of each suspension was used to prepare an interface, according to the protocol<br />
#200x diluted GFP solution was used to prepare the emulsion<br />
*Additionally, the emulsion prepared the[[Imperial/Wet Lab/Lab Notebook/2007-08-16#Pilot Preparation of Vesicles|day before]] (1-day old emulsion) was stirred once again to see if it would yield vesicles.<br />
<br />
#Another sample from the vesicles prepared the [[Imperial/Wet Lab/Lab Notebook/2007-08-16|day before]] was taken for observation under the fluorescence microscope<br />
#200x diluted GFP solution was also observed under the fluorescence microscope<br />
#*It was found that the GFP solution was too weak to be observed in the microscope, and therefore the emulsions being prepared would not work.<br />
#*It was also found that the GFP solution needs to be protected from light in order to avoid rapid degradation. <br />
#Upon these discoveries, 10x diluted GFP was added to the three emulsions above while they were already being mixed with the 200x diluted GFP.<br />
<br />
*Six samples were prepared:<br />
#Three following the protocol, with 2ml suspension interface and 100&mu;l of each of three emulsions added:<br />
#*Sample 1: Well desiccated suspension interface with 100&mu;l of emulsion from well desiccated suspension.<br />
#*Sample 2: Poorly desiccated suspension interface with 100&mu;l of emulsion from Poorly desiccated suspension.<br />
#*Sample 3: Poorly desiccated suspension interface with 100&mu;l of 1-day old emulsion.<br />
#Three using 2ml of emulsion to form the interface, without further addition of material:<br />
#*Sample 4: 2ml of emulsion from well desiccated suspension.<br />
#*Sample 5: 2ml of emulsion from poorly desiccated suspension.<br />
#*Sample 6: 2ml of 1-day old emulsion.<br />
<br />
*'''Results''': A fluorescence microscope was used to inspect the samples:<br />
**Sample 1: Tiny and rare specks of fluorescence were seen, only under 100x magnification, but they did not look like vesicles.<br />
**Sample 2: Sample not looked at.<br />
**Sample 3: Sample not looked at.<br />
**Sample 4: One fluorescent vesicle was observed, under 100x magnification, but the GFP was aggregated on the membrane (a green circle, not a green dot).<br />
**Sample 5: Some GFP aggregates were seen under 40x and 100x magnification, but no structures resembling vesicles.<br />
**Sample 6: Sample not looked at.<br />
<br />
<br=all clear><br />
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{{Template:IC07labnotebook}}</div>Dirkvshttp://2007.igem.org/wiki/index.php/Imperial/Dry_Lab/Data_AnalysisImperial/Dry Lab/Data Analysis2007-10-26T22:49:51Z<p>Dirkvs: /* Principle of method of parameter extraction */</p>
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<div>{{Template:IC07navmenu}}<br />
<br clear="all"><br />
__NOTOC__<br />
=Data Analysis=<br />
<br />
==Introduction==<br />
Data analysis involves manipulating experimental data with the objective of extracting useful information. This then allows us to test our original hypotheses surrounding the problem, and in doing so, test the stringency/validity of our representative model.<br />
<br />
[[Image: IC07_LeastSqr.gif|thumb|left|450px| '''Fig. 1''': Curve/Shape-fitting]]<br />
If the model proves to be valid, data analysis likewise provides a means of parameter extraction essential in rendering our theoretical model more realistic (as it gleans parameters from actual expimental data).<br />
<br />
Our approach to data analysis utilizes curve/shape-fitting by non-linear regression (employing the least-squares method).<br><br><br />
<br />
==Principal of method of parameter extraction==<br />
[[Image: IC07_nonLinearLSqr.gif|thumb|right|450px|Non-linear least-squares curve-fitting]]<br />
The method uses weighted non-linear leasts-squares. This technique involves obtaining the best-fitting non-linear curve for a given set of parameters from the parameter space. This procedure involves minimizing the sum of the squares of the offsets from the chosen curve. [http://mathworld.wolfram.com/LeastSquaresFitting.html] Here, the offsets refering to the difference between the chosen non-linear curve and the experimental data, at a particular value of the independent variable. <br />
<br />
A weighted non-linear least-squares is used, so that that the integrity of the data does not corrupt the extracted parameters. Weightings are assigned to adjacent experimental data points, according to their variance from the general trend/behaviour.<br />
<br />
====Representative example====<br />
Consider the following model<br />
<br />
==Our Model==</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/mainpage.cssUser:Dirkvs/Stylesheets/mainpage.css2007-10-26T22:48:31Z<p>Dirkvs: </p>
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min-height: 1000px;<br />
padding-left: 17px;<br />
color: #666;<br />
font-size: 11pt;<br />
}<br />
<br />
#maincontent h2 {<br />
margin: 0;<br />
}<br />
<br />
#maincontent a {<br />
text-decoration: none;<br />
color: #0a5b86;<br />
font-family: "Helvetica", Verdana, arial, sans-serif;<br />
}<br />
<br />
#maincontent a:hover {<br />
color: #7395ba;<br />
}<br />
<br />
<br />
.metainfo {<br />
font-size: 1.1em;<br />
width: 100%;<br />
position: relative;<br />
top: 0.5em;<br />
left: 1em;<br />
margin: 0 0 0 0;<br />
}<br />
<br />
.metainfo h2 {<br />
display: inline;<br />
font-size: 14pt;<br />
}<br />
<br />
.metainfo span {<br />
position: absolute;<br />
right: 0px;<br />
bottom: 0px;<br />
color: #454545;<br />
}<br />
<br />
#OverviewBox {<br />
margin: 14px 3px 14px;<br />
background-color: #d9f0f8;<br />
border-right: 4px solid #ade2f2;<br />
border-bottom: 4px solid #ade2f2;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
#OverviewBox h2 {<br />
padding:10px 0 0 10px;<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
<br />
#FeatureBox {<br />
margin: 14px 3px 14px;<br />
background-color: #f5facf;<br />
border-right: 4px solid #eaf7a6;<br />
border-bottom: 4px solid #eaf7a6;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
#SummaryBox {<br />
margin: 10px 7px 10px 5px;<br />
background-color: #eeeeee;<br />
border-right: 5px solid #d9dddd;<br />
border-bottom: 5px solid #d9dddd;<br />
font-size: 1em;<br />
min-height: 50px;<br />
width: 250px;<br />
}<br />
<br />
#SummaryBox h2 {<br />
padding:10px 0 0 10px;<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
<br />
.parabox {<br />
width: 230px;<br />
margin:1em;<br />
font-size: 1em;<br />
}<br />
<br />
#TechnoBox {<br />
margin: 14px 3px 14px;<br />
background-color: #dff7d6;<br />
border-right: 4px solid #b8efb0;<br />
border-bottom: 4px solid #b8efb0;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
#newscol {<br />
position: relative;<br />
float: left;<br />
width: 216px;<br />
margin-left: 16px;<br />
clear:left;<br />
}<br />
<br />
.newsmeta {<br />
border-bottom-color: #d2eef3;<br />
border-bottom-width: 1px;<br />
border-bottom-style: solid;<br />
width: 100%;<br />
margin: 0 0 20px 0;<br />
clear:both; <br />
}<br />
<br />
.newsmeta h2 {<br />
padding-bottom: 0px;<br />
font-size: 1em;<br />
margin:0 0;<br />
}<br />
<br />
.blurb {<br />
width: 580px;<br />
margin:8px 0 4px 0;<br />
padding-right: 1em; <br />
padding-left: 1em; <br />
text-align: justify;<br />
font: normal 1.4em georgia, serif;<br />
color: #555555;<br />
line-height: 1.2em;<br />
}<br />
<br />
#maincol {<br />
width: 600px;<br />
margin: 0 16px 16px 8px;<br />
padding: 0px 0 8px 0px;<br />
}<br />
<br />
#maincol p.main {<br />
margin: 0 8px 0 8px;<br />
line-height: 1.4em;<br />
font-size: .9em;<br />
color: 666666;<br />
margin: 6px 8px 0 8px;<br />
font-family: Georgia, "Helvetica", Times, serif;<br />
text-align: justify;<br />
}<br />
<br />
#maincol h2.main {<br />
margin: 20px 0 4px 0;<br />
font-size: 1.2em;<br />
}<br />
<br />
#sidebar {<br />
left: 630px;<br />
top: 160px;<br />
position: absolute;<br />
height: 1000px;<br />
width: 180px;<br />
background-position: left top;<br />
background-image: #ffffff;<br />
font-family: "Lucida Grande", Verdana, Arial, sans-serif;<br />
color: #666666;<br />
font-size: 0.9em;<br />
}<br />
<br />
a {<br />
color: #0a5b86;<br />
text-decoration:none;<br />
}<br />
<br />
a:hover {<br />
color: #7395ba;<br />
}<br />
<br />
#sidebar h2 {<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
<br />
#sidebar li a { color: #0a5b86;}<br />
<br />
.post-it {<br />
position: relative;<br />
top: 10px;<br />
float: left;<br />
margin-right: 16px;<br />
margin-bottom: 10px;<br />
padding: 12px 8px 8px 8px;<br />
width: 236px;<br />
font-size: .9em;<br />
line-height: 1.3em;<br />
text-align: justify;<br />
clear:both;<br />
filter:alpha(opacity=85);<br />
-moz-opacity:0.85;<br />
}<br />
<br />
.calbox {<br />
width: 286px;<br />
padding: 12px 0px 0px 8px;<br />
filter:alpha(opacity=65);<br />
-moz-opacity:0.65;<br />
}<br />
<br />
.bluebox {<br />
background-color: #d9f0f8;<br />
border-right: 12px solid #ade2f2;<br />
}<br />
<br />
.yellowbox {<br />
background-color: #f7fbd7;<br />
border-right: 12px solid #eef8b3;<br />
}<br />
<br />
.pinkbox {<br />
background-color: #f0dbcc;<br />
border-right: 12px solid #ddb99e;<br />
}<br />
<br />
.greenbox {<br />
background-color: #dff7d6;<br />
border-right: 12px solid #b8efb0;<br />
}<br />
<br />
.whitebox {<br />
background-color: #eeeeee;<br />
border-right: 12px solid #d9dddd;<br />
}</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/mainpage.cssUser:Dirkvs/Stylesheets/mainpage.css2007-10-26T22:41:37Z<p>Dirkvs: </p>
<hr />
<div>#content{ padding: 0; }<br />
<br />
#wrap { <br />
/* width: 945px; */<br />
margin-left: -1em;<br />
color: #666;<br />
}<br />
<br />
#wrap a {<br />
color: #0a5b86;<br />
text-decoration:none;<br />
}<br />
<br />
#wrap a:hover {<br />
color: #0a5b86;<br />
}<br />
<br />
#wrap a img{<br />
border:0;<br />
}<br />
<br />
#wrap h2 {<br />
margin:0 0 10px 0;<br />
font-family: "Lucida Grande", Verdana, Arial, sans-serif;<br />
font-size: 1.25em;<br />
font-style: bold;<br />
border-bottom: none;<br />
}<br />
<br />
#wrap p { margin: 0;}<br />
<br />
#maincontent {<br />
width: 800px;<br />
min-height: 1000px;<br />
padding-left: 17px;<br />
color: #666;<br />
font-size: 11pt;<br />
}<br />
<br />
#maincontent h2 {<br />
margin: 0;<br />
}<br />
<br />
#maincontent a {<br />
text-decoration: none;<br />
color: #0a5b86;<br />
font-family: "Helvetica", Verdana, arial, sans-serif;<br />
}<br />
<br />
#maincontent a:hover {<br />
color: #7395ba;<br />
}<br />
<br />
<br />
.metainfo {<br />
font-size: 1.1em;<br />
width: 100%;<br />
position: relative;<br />
top: 0.5em;<br />
left: 1em;<br />
margin: 0 0 0 0;<br />
}<br />
<br />
.metainfo h2 {<br />
display: inline;<br />
font-size: 14pt;<br />
}<br />
<br />
.metainfo span {<br />
position: absolute;<br />
right: 0px;<br />
bottom: 0px;<br />
color: #454545;<br />
}<br />
<br />
#OverviewBox {<br />
margin: 14px 3px 14px;<br />
background-color: #d9f0f8;<br />
border-right: 4px solid #ade2f2;<br />
border-bottom: 4px solid #ade2f2;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
#OverviewBox h2 {<br />
padding:10px 0 0 10px;<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
<br />
#FeatureBox {<br />
margin: 14px 3px 14px;<br />
background-color: #f5facf;<br />
border-right: 4px solid #eaf7a6;<br />
border-bottom: 4px solid #eaf7a6;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
#SummaryBox {<br />
margin: 10px 7px 10px 5px;<br />
background-color: #eeeeee;<br />
border-right: 5px solid #d9dddd;<br />
border-bottom: 5px solid #d9dddd;<br />
font-size: 1em;<br />
min-height: 50px;<br />
width: 250px;<br />
}<br />
<br />
#SummaryBox h2 {<br />
padding:10px 0 0 10px;<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
<br />
.parabox {<br />
width: 230px;<br />
margin:1em;<br />
font-size: 1em;<br />
}<br />
<br />
#TechnoBox {<br />
margin: 14px 3px 14px;<br />
background-color: #dff7d6;<br />
border-right: 4px solid #b8efb0;<br />
border-bottom: 4px solid #b8efb0;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
#newscol {<br />
position: relative;<br />
float: left;<br />
width: 216px;<br />
margin-left: 16px;<br />
clear:left;<br />
}<br />
<br />
.newsmeta {<br />
border-bottom-color: #d2eef3;<br />
border-bottom-width: 1px;<br />
border-bottom-style: solid;<br />
width: 100%;<br />
margin: 0 0 20px 0;<br />
clear:both; <br />
}<br />
<br />
.newsmeta h2 {<br />
padding-bottom: 0px;<br />
font-size: 1em;<br />
margin:0 0;<br />
}<br />
<br />
.blurb {<br />
width: 580px;<br />
margin:8px 0 4px 0;<br />
padding-right: 1em; <br />
padding-left: 1em; <br />
text-align: justify;<br />
font: normal 1.4em georgia, serif;<br />
color: #555555;<br />
line-height: 1.2em;<br />
}<br />
<br />
#maincol {<br />
width: 600px;<br />
margin: 0 16px 16px 8px;<br />
padding: 0px 0 8px 0px;<br />
}<br />
<br />
#maincol p.main {<br />
margin: 0 8px 0 8px;<br />
line-height: 1.4em;<br />
font-size: .9em;<br />
color: 666666;<br />
margin: 6px 8px 0 8px;<br />
font-family: Georgia, "Helvetica", Times, serif;<br />
text-align: justify;<br />
}<br />
<br />
#maincol h2.main {<br />
margin: 20px 0 4px 0;<br />
font-size: 1.2em;<br />
}<br />
<br />
#sidebar {<br />
left: 630px;<br />
top: 160px;<br />
position: absolute;<br />
height: 1000px;<br />
width: 180px;<br />
background-position: left top;<br />
background-image: #ffffff;<br />
font-family: "Lucida Grande", Verdana, Arial, sans-serif;<br />
color: #666666;<br />
font-size: 0.9em;<br />
}<br />
<br />
a {<br />
color: #0a5b86;<br />
text-decoration:none;<br />
}<br />
<br />
a:hover {<br />
color: #7395ba;<br />
}<br />
<br />
#sidebar h2 {<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
<br />
#sidebar li a { color: #0a5b86;}<br />
<br />
.post-it {<br />
position: relative;<br />
top: 0px;<br />
float: left;<br />
margin-right: 16px;<br />
margin-bottom: 10px;<br />
padding: 0px 8px 8px 8px;<br />
width: 236px;<br />
font-size: .9em;<br />
line-height: 1.3em;<br />
text-align: justify;<br />
clear:both;<br />
filter:alpha(opacity=85);<br />
-moz-opacity:0.85;<br />
}<br />
<br />
.calbox {<br />
width: 286px;<br />
padding: 12px 0px 0px 8px;<br />
filter:alpha(opacity=65);<br />
-moz-opacity:0.65;<br />
}<br />
<br />
.bluebox {<br />
background-color: #d9f0f8;<br />
border-right: 12px solid #ade2f2;<br />
}<br />
<br />
.yellowbox {<br />
background-color: #f7fbd7;<br />
border-right: 12px solid #eef8b3;<br />
}<br />
<br />
.pinkbox {<br />
background-color: #f0dbcc;<br />
border-right: 12px solid #ddb99e;<br />
}<br />
<br />
.greenbox {<br />
background-color: #dff7d6;<br />
border-right: 12px solid #b8efb0;<br />
}<br />
<br />
.whitebox {<br />
background-color: #eeeeee;<br />
border-right: 12px solid #d9dddd;<br />
}</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/mainpage.cssUser:Dirkvs/Stylesheets/mainpage.css2007-10-26T22:40:49Z<p>Dirkvs: </p>
<hr />
<div>#content{ padding: 0; }<br />
<br />
#wrap { <br />
/* width: 945px; */<br />
margin-left: -1em;<br />
color: #666;<br />
}<br />
<br />
#wrap a {<br />
color: #0a5b86;<br />
text-decoration:none;<br />
}<br />
<br />
#wrap a:hover {<br />
color: #0a5b86;<br />
}<br />
<br />
#wrap a img{<br />
border:0;<br />
}<br />
<br />
#wrap h2 {<br />
margin:0 0 10px 0;<br />
font-family: "Lucida Grande", Verdana, Arial, sans-serif;<br />
font-size: 1.25em;<br />
font-style: bold;<br />
border-bottom: none;<br />
}<br />
<br />
#wrap p { margin: 0;}<br />
<br />
#maincontent {<br />
width: 800px;<br />
min-height: 1000px;<br />
padding-left: 17px;<br />
color: #666;<br />
font-size: 11pt;<br />
}<br />
<br />
#maincontent h2 {<br />
margin: 0;<br />
}<br />
<br />
#maincontent a {<br />
text-decoration: none;<br />
color: #0a5b86;<br />
font-family: "Helvetica", Verdana, arial, sans-serif;<br />
}<br />
<br />
#maincontent a:hover {<br />
color: #7395ba;<br />
}<br />
<br />
<br />
.metainfo {<br />
font-size: 1.1em;<br />
width: 100%;<br />
position: relative;<br />
top: 0.5em;<br />
left: 1em;<br />
margin: 0 0 0 0;<br />
}<br />
<br />
.metainfo h2 {<br />
display: inline;<br />
font-size: 14pt;<br />
}<br />
<br />
.metainfo span {<br />
position: absolute;<br />
right: 0px;<br />
bottom: 0px;<br />
color: #454545;<br />
}<br />
<br />
#OverviewBox {<br />
margin: 14px 3px 14px;<br />
background-color: #d9f0f8;<br />
border-right: 4px solid #ade2f2;<br />
border-bottom: 4px solid #ade2f2;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
#OverviewBox h2 {<br />
padding:10px 0 0 10px;<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
<br />
#FeatureBox {<br />
margin: 14px 3px 14px;<br />
background-color: #f5facf;<br />
border-right: 4px solid #eaf7a6;<br />
border-bottom: 4px solid #eaf7a6;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
#SummaryBox {<br />
margin: 10px 7px 10px 5px;<br />
background-color: #eeeeee;<br />
border-right: 5px solid #d9dddd;<br />
border-bottom: 5px solid #d9dddd;<br />
font-size: 1em;<br />
min-height: 50px;<br />
width: 250px;<br />
}<br />
<br />
#SummaryBox h2 {<br />
padding:10px 0 0 10px;<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
<br />
.parabox {<br />
width: 230px;<br />
margin:1em;<br />
font-size: 1em;<br />
}<br />
<br />
#TechnoBox {<br />
margin: 14px 3px 14px;<br />
background-color: #dff7d6;<br />
border-right: 4px solid #b8efb0;<br />
border-bottom: 4px solid #b8efb0;<br />
font-size: 0.9em;<br />
min-height: 100px;<br />
width: 600px;<br />
}<br />
<br />
.contentbox {<br />
width: 580px;<br />
margin:1em;<br />
}<br />
<br />
#newscol {<br />
position: relative;<br />
float: left;<br />
width: 216px;<br />
margin-left: 16px;<br />
clear:left;<br />
}<br />
<br />
.newsmeta {<br />
border-bottom-color: #d2eef3;<br />
border-bottom-width: 1px;<br />
border-bottom-style: solid;<br />
width: 100%;<br />
margin: 0 0 20px 0;<br />
clear:both; <br />
}<br />
<br />
.newsmeta h2 {<br />
padding-bottom: 0px;<br />
font-size: 1em;<br />
margin:0 0;<br />
}<br />
<br />
.blurb {<br />
width: 580px;<br />
margin:8px 0 4px 0;<br />
padding-right: 1em; <br />
padding-left: 1em; <br />
text-align: justify;<br />
font: normal 1.4em georgia, serif;<br />
color: #555555;<br />
line-height: 1.2em;<br />
}<br />
<br />
#maincol {<br />
width: 600px;<br />
margin: 0 16px 16px 8px;<br />
padding: 0px 0 8px 0px;<br />
}<br />
<br />
#maincol p.main {<br />
margin: 0 8px 0 8px;<br />
line-height: 1.4em;<br />
font-size: .9em;<br />
color: 666666;<br />
margin: 6px 8px 0 8px;<br />
font-family: Georgia, "Helvetica", Times, serif;<br />
text-align: justify;<br />
}<br />
<br />
#maincol h2.main {<br />
margin: 20px 0 4px 0;<br />
font-size: 1.2em;<br />
}<br />
<br />
#sidebar {<br />
left: 630px;<br />
top: 160px;<br />
position: absolute;<br />
height: 1000px;<br />
width: 180px;<br />
background-position: left top;<br />
background-image: #ffffff;<br />
font-family: "Lucida Grande", Verdana, Arial, sans-serif;<br />
color: #666666;<br />
font-size: 0.9em;<br />
}<br />
<br />
a {<br />
color: #0a5b86;<br />
text-decoration:none;<br />
}<br />
<br />
a:hover {<br />
color: #7395ba;<br />
}<br />
<br />
#sidebar h2 {<br />
border-bottom: 1px solid;<br />
border-bottom-color: #b7b7b7;<br />
}<br />
<br />
#sidebar li a { color: #0a5b86;}<br />
<br />
.post-it {<br />
position: relative;<br />
top: 0px;<br />
float: left;<br />
margin-right: 16px;<br />
margin-bottom: 10px;<br />
padding: 12px 8px 8px 8px;<br />
width: 236px;<br />
font-size: .9em;<br />
line-height: 1.3em;<br />
text-align: justify;<br />
clear:both;<br />
filter:alpha(opacity=85);<br />
-moz-opacity:0.85;<br />
}<br />
<br />
.calbox {<br />
width: 286px;<br />
padding: 12px 0px 0px 8px;<br />
filter:alpha(opacity=65);<br />
-moz-opacity:0.65;<br />
}<br />
<br />
.bluebox {<br />
background-color: #d9f0f8;<br />
border-right: 12px solid #ade2f2;<br />
}<br />
<br />
.yellowbox {<br />
background-color: #f7fbd7;<br />
border-right: 12px solid #eef8b3;<br />
}<br />
<br />
.pinkbox {<br />
background-color: #f0dbcc;<br />
border-right: 12px solid #ddb99e;<br />
}<br />
<br />
.greenbox {<br />
background-color: #dff7d6;<br />
border-right: 12px solid #b8efb0;<br />
}<br />
<br />
.whitebox {<br />
background-color: #eeeeee;<br />
border-right: 12px solid #d9dddd;<br />
}</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/mainpage.cssUser:Dirkvs/Stylesheets/mainpage.css2007-10-26T22:39:01Z<p>Dirkvs: </p>
<hr />
<div>#content{ padding: 0; }<br />
<br />
#wrap { <br />
/* width: 945px; */<br />
margin-left: -1em;<br />
color: #666;<br />
}<br />
<br />
#wrap a {<br />
color: #0a5b86;<br />
text-decoration:none;<br />
}<br />
<br />
#wrap a:hover {<br />
color: #0a5b86;<br />
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__NOTOC__<br />
= Infector Detector: Introduction =<br />
<br />
[[Image:IC07 cartoon.png|center|900px]] <br clear="all"><br />
<br />
Infector Detector tackles the ongoing problem of catheter-associated urinary tract infections. To do this, we looked at how infections develop - as biofilms - and designed a system which would be able to detect their presence. We have created a system which is capable of detecting one of the types of signalling molecules found in biofilms, AHL, and visibly report its presence by producing a fluorescent protein.<br />
<br />
== Motivation ==<br />
<br />
=== Urinary Catheter Infections ===<br />
'''Urinary catheters''' are the major source of '''hospital infections''' in the US: more than 40%, or '''1 million''' infections per year '''costing''' health services up to '''1 billion USD'''<sup>[[#References |1-4]]</sup>. It is a very uncomfortable and painful condition. Infection develops in up to '''25% of patients''' requiring a urinary catheter for more than seven days<sup>[[#References |5-6]]</sup>. They are the second most common cause of hospital bloodstream infection<sup>[[#References |8-10]]</sup>. Additionally, urinary catheters comprise the largest institutional reservoir of nosocomial '''antibiotic-resistant pathogens'''.<sup>[[#References |5-10]]</sup><br />
<br />
E.coli alone is responsible for over a quarter of urinary catheter infections. Pseudomonas aeruginosa is another major agent. Such infections are caused by a bacterial biofilm that forms on the surface of the catheter, and then spreads up into the urethra. <br />
<br />
{| style="v-align: top;"<br />
|-<br />
|[[image:IC07_catheter.gif|thumb|right|300px|Male and female urinary catheters. [http://www.oldcity.org.uk/cauda_equina/images/catheter.gif www.oldcity.org.uk]]] || [[Image:IC07 infectedcatheter.jpg|thumb|An infected catheter<sup>[[#References |12]]</sup>.]] ||[[Image:IC07 infectedcatheter2.jpg|thumb|240px|Scanning electron micrograph of the surface of a catheter removed from patient with catheter-associated UTI<sup>[[#References |11]]</sup>.]]<br />
|}<br />
<br />
<br clear="all"><br />
<br />
[[Image:IC07 biofilmspread.jpg|thumb|400px|right|Biofilm formation and spread: '''1''' Cells attach to a surface. '''2''' Cells begin producing polysaccharides. '''3''' A protective layer forms around the cells. '''4''' The biofilm grows. '''5''' Cells are released into the environment, continuing the cycle.]]<br />
<br />
=== Biofilms ===<br />
<br />
A biofilm forms when an organism attaches to a surface, and begins producing polysaccharides. This forms a protective matrix around the organism, to which other cells can attach and secrete even more polysaccharides. The biofilm grows until it begins to release cells and other debris into its environment. Released cells then attach to other parts of the surface, starting the cycle anew<sup>[[#References |13-15]]</sup>.<br />
<br />
And like that, the biofilm spreads around and creeps along a surface. This process takes place between a few hours to a few days.<br />
<br />
Biofilms are very tough to eradicate - they are difficult to clean, and cells contained within are very hard to target with normal antibiotics or antiseptics.<br />
<br />
<br clear="all"><br />
<br />
=== Cell signalling inside biofilms ===<br />
<br />
The behaviour of cells in a biofilm is very different from that of those in planktonic state. They talk to each other through molecular signalling mechanisms, such as quorum sensing and wall sensing. An important characteristic of biofilms, then, is that there are signal molecules floating around inside of it.<br />
<br />
[[Image:IC07 AHLmechanism.jpg|thumb|left|400px|The AHL quorum sensing mechanism. AHL enters the cell, where it binds to LuxR, forming a complex capable of activating the pLux promoter.]]<br />
<br />
One type of signalling molecule, AHL, is produced by Pseudomonas aeruginosa when forming biofilms<sup>[[#References |16]]</sup>. MRSA has a similar behaviour – the agr quorum sensing mechanism<sup>[[#References |17]]</sup>. Every biofilm has a similar mechanism, because this type of signalling controls the growth of the biofilm.<br />
<br />
Our system, Infector Detector, exploits exactly this mechanism in order to detect biofilms.<br />
<br />
<br clear="all"><br />
<br />
== Achievements ==<br />
* We used synthetic biology to tackle the problem of urinary catheter infections. We achieved this using only parts available in the Registry, and a new in-vitro chassis.<br />
* We designed, built, and tested successfully a system capable of detecting AHL at concentrations as low as 5nM, and responding within 3 hours.<br />
* We tested the system in-vivo, using E.coli, and in-vitro, using titrated AHL. The next step is to test it on real biofilms.<br />
<br />
<br clear="all"><br />
<br />
<center> On to next stage : | [https://2007.igem.org/Imperial/Infector_Detector/Specification Specifications >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# Stamm WE. Catheter-associated urinary tract infections: Epidemiology, pathogenesis, and prevention. Am J Med 1991;91(Suppl 3B):65S-71S. <br />
# Burke JP, Riley DK. Nosocomial urinary tract infection. In: Mayhall CG, editor. Hospital epidemiology and infection control. Baltimore: Williams and Wilkins; 1996. p. 139-53. <br />
# Warren JW. Catheter-associated urinary tract infections. Infect Dis Clin North Am 1997;11:609-22. <br />
# Kunin CM. Care of the urinary catheter. In: Urinary tract infections: detection, prevention and management. Fifth ed. Baltimore: Williams and Wilkins; 1997. p. 227-99. <br />
# Kunin CM, McCormack RC. Prevention of catheter-induced urinary-tract infections by sterile closed drainage. N Engl J Med 1966;274:1155-61. <br />
# Garibaldi RA, et al. An evaluation of daily bacteriologic monitoring to identify preventable episodes of catheter associated UTI. Infect Control 1982;3:466-70. <br />
# Stark RP, Maki DG. Bacteriuria in the catheterized patient. N Engl J Med 1984;311:560-4. <br />
# Maki DG. Nosocomial bacteremia. An epidemiologic overview. Am J Med 1981;70:719-32. <br />
# Krieger JN, Kaiser DIL, Wenzel RP. Urinary tract etiology of bloodstream infections in hospitalized patients. J Infect Dis 1983;148:57-62. <br />
# Bryan CS, Reynolds KL. Hospital-acquired bacteremic urinary tract infection: epidemiology and outcome. J Urol 1984,132:494-8. <br />
# [http://www.medscape.com/viewarticle/416647_3 Bacterial Biofilms in Urology].Infect Urol 11(6):169-175, 1998.<br />
# Laube, N. Diamonds are a Urologist's Best Friend. [http://www.biofilmsonline.com/cgi-bin/biofilmsonline/00271.html BiofilmsOnline.com] 18-Nov-2004.<br />
# Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis 2002 Sep; 8(9) 881-90. pmid:12194761. PubMed HubMed [biofilm4]<br />
# Donlan RM. Biofilm formation: a clinically relevant microbiological process. Clin Infect Dis 2001 Oct 15; 33(8) 1387-92.<br />
# Tenke P, Riedl CR, Jones GL, Williams GJ, Stickler D, and Nagy E. Bacterial biofilm formation on urologic devices and heparin coating as preventive strategy. Int J Antimicrob Agents 2004 Mar; 23 Suppl 1 S67-74. doi:10.1016/j.ijantimicag.2003.12.007 <br />
# David J. Stickler, et al. Biofilms on Indwelling Urethral Catheters Produce Quorum-Sensing Signal Molecules In Situ and In Vitro. Applied and Environmental Microbiology, September 1998, p. 3486-3490, Vol. 64, No. 9 <br />
# Manago K, et al. Biofilm formation by and accessory gene regulator typing of methicillin-resistant Staphylococcus aureus strains recovered from patients with nosocomial infections. Infect Control Hosp Epidemiol 2006 Feb; 27(2) 188-90. doi:10.1086/500620 pmid:16465637.</div>Dirkvshttp://2007.igem.org/wiki/index.php/Template:IC07navmenuTemplate:IC07navmenu2007-10-26T22:27:17Z<p>Dirkvs: </p>
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__NOTOC__<br />
= Infector Detector: Design =<br />
<br />
<center>[[Image:IC07 design ID-bb1.png]] </center><br />
<br><br />
<br />
{|border="1"<br />
|-<br />
|width="300px"|'''Specification'''<br />
|width="420px"|'''Design Solution'''<br />
|width="100px"|'''System Level'''<br />
|- style="background:#eeffee"<br />
|'''Health & Safety''' - System must not contain living cells, or cause any harm to patient.<br />
|Use a Cell Free System e.g. Promega's S30 Cell Extract<br />
|Chassis<br />
|- style="background:#eeffee"<br />
|'''Shelf-life''' - System must have a shelf life of 7 days.<br />
|Use a Cell Free System that can be packaged and stored in a -80&deg;C freezer for 7 days.<br />
|Chassis<br />
|- style="background:#eeffee"<br />
|'''Application''' - System must be applied as a cream or spray.<br />
|Package the system in liquid form.<br />
|Chassis<br />
|- style="background:#eeeeff"<br />
|'''Inputs''' - System must be sensitive to AHL concentration between 5-50nM.<br />
|Sensitive AHL receiver to detect low AHL levels - Part no. F2620.<br />
|Construct<br />
|- style="background:#eeeeff"<br />
|'''Outputs''' - System must give a visual signal if AHL is present.<br />
|Couple AHL to a reporter system expressing fluoresent protein eg. RFP<br />
|Construct<br />
|- style="background:#ffeeee"<br />
|'''Operating Conditions''' - System must operate within temperature 20&deg;-30&deg;C.<br />
|Gene expression systems and protein must be thermostable. Characterise a Cell Free System at these temperatures.<br />
|Construct & Chassis<br />
|- style="background:#ffeeee"<br />
|'''Response Time''' - System needs to have a response time under 3 hours.<br />
|To be Determined - this is hard to design for. Protease Inhibitor of Cell Extract should ensure degradation of Visual Reporter is minimal.<br />
|Construct & Chassis<br />
|}<br />
<br clear="all"><br />
<br />
==Chassis Selection==<br />
[[Image:IC07_CFS_components.png|thumb|left|340px|Commercial S30 ''E. coli'' Cell Extract in bulk solution + packaging to last 7 days]]<br />
<br />
We have chosen to use the commercially available S30 ''E. coli'' cell extract made by Promega. After looking into a variety of different [[Imperial/Cell-Free/Whatis|cell-free systems]], we decided that this chassis is most suitable. In particular, it allows us to comply with the Health and Safety regulations of the field we are working in, as we do not want replicative bacteria that could potentially be pathogenic to come in contact with urinary catheters.<br />
<br />
In addition to complying with health regulations, the S30 cell extract is commercially available, meaning that it has been shown to work. <sup>[[#References|1]]</sup> This allows our focus to be on tuning the chassis to suit our needs, rather than trying to make the chassis work in the first place.<br />
<br />
<br clear="all"><br />
<br />
<br />
== DNA Constructs ==<br />
<br />
'''Concept 1'''<br /> <center>[[Image:IC07_design_ID.png|550px]]</center><br />
<br />
Our aim was to create a system that can quickly detect a small (5nM) concentration of AHL in solution. We needed a well characterized detector system sensitive to 3OC6HSL which we will attach to a standard GFP reporter to determine its response to GFP. The first construct is appealing as the AHL receiver in front is well-documented as part [http://partsregistry.org/Part:BBa_F2620 BBa_F2620], thus giving more assurance in the reproducible nature of our results.<br />
<br />
<br />
'''Concept 2'''<br /> <center>[[Image:IC07_design_ID-con2.png|550px]]</center><br />
<br />
<br />
However, the weakness of concept 1 lies in the fact that it does not respond uniformly to constant AHL concentration since the activator protein LuxR is produced by a promoter and not maintained at a constant level. This makes it hard to relate the output of the system to the AHL input. However, if purified LuxR could be added to the system instead, the construct could be simplified by removing the constitutive LuxR production. This would give us more control of the input, and therefore better reproducibility.<br />
<br />
<br clear="all"><br />
<br />
<br />
<br />
<center> [https://2007.igem.org/Imperial/Infector_Detector/Specification << Specification] | Design | [https://2007.igem.org/Imperial/Infector_Detector/Modelling Modelling >>]<br />
</center><br />
<br />
== References ==<br />
<br />
# [https://promega.de/tbs/tb092/tb092.pdf Promega S30 Cell Extract documentation]</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/IC07persist.cssUser:Dirkvs/Stylesheets/IC07persist.css2007-10-26T22:25:37Z<p>Dirkvs: </p>
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#tabs a.current:hover span {<br />
background-position:100% 0px;<br />
}</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/IC07persist.cssUser:Dirkvs/Stylesheets/IC07persist.css2007-10-26T22:20:56Z<p>Dirkvs: </p>
<hr />
<div>#tabs {<br />
float:left; <br />
position:absolute;<br />
left60px; <br />
width:100%;<br />
background:#ffffff;<br />
font-size:100%;<br />
font-weight:bold;<br />
line-height:normal;<br />
border-bottom:1px solid #0a5b86; <br />
}<br />
<br />
#tabs ul {<br />
margin:0;<br />
padding:10px 10px 0 10px;<br />
list-style:none;<br />
}<br />
<br />
#tabs li {<br />
display:inline;<br />
margin:0;<br />
padding:0;<br />
}<br />
<br />
#tabs a {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/06/IC07_tableft.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/8/87/IC07_tabright.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/03/IC07_tableftactive.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a.current span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/4/4d/IC07_tabrightactive.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover {<br />
background-position:0% -42px;<br />
}<br />
<br />
#tabs a:hover span {<br />
background-position:100% -42px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current:hover {<br />
background-position:0% 0px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
background-position:100% 0px;<br />
}</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/IC07persist.cssUser:Dirkvs/Stylesheets/IC07persist.css2007-10-26T22:20:28Z<p>Dirkvs: </p>
<hr />
<div>#tabs {<br />
float:left; <br />
position:relative;<br />
left60px; <br />
width:100%;<br />
background:#ffffff;<br />
font-size:100%;<br />
font-weight:bold;<br />
line-height:normal;<br />
border-bottom:1px solid #0a5b86; <br />
}<br />
<br />
#tabs ul {<br />
margin:0;<br />
padding:10px 10px 0 10px;<br />
list-style:none;<br />
}<br />
<br />
#tabs li {<br />
display:inline;<br />
margin:0;<br />
padding:0;<br />
}<br />
<br />
#tabs a {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/06/IC07_tableft.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/8/87/IC07_tabright.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/03/IC07_tableftactive.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a.current span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/4/4d/IC07_tabrightactive.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover {<br />
background-position:0% -42px;<br />
}<br />
<br />
#tabs a:hover span {<br />
background-position:100% -42px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current:hover {<br />
background-position:0% 0px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
background-position:100% 0px;<br />
}</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/IC07persist.cssUser:Dirkvs/Stylesheets/IC07persist.css2007-10-26T22:19:58Z<p>Dirkvs: </p>
<hr />
<div>#tabs {<br />
float:right; <br />
position:relative;<br />
width:100%;<br />
background:#ffffff;<br />
font-size:100%;<br />
font-weight:bold;<br />
line-height:normal;<br />
border-bottom:1px solid #0a5b86; <br />
}<br />
<br />
#tabs ul {<br />
margin:0;<br />
padding:10px 10px 0 10px;<br />
list-style:none;<br />
}<br />
<br />
#tabs li {<br />
display:inline;<br />
margin:0;<br />
padding:0;<br />
}<br />
<br />
#tabs a {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/06/IC07_tableft.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/8/87/IC07_tabright.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/03/IC07_tableftactive.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a.current span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/4/4d/IC07_tabrightactive.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover {<br />
background-position:0% -42px;<br />
}<br />
<br />
#tabs a:hover span {<br />
background-position:100% -42px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current:hover {<br />
background-position:0% 0px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
background-position:100% 0px;<br />
}</div>Dirkvshttp://2007.igem.org/wiki/index.php/User:Dirkvs/Stylesheets/IC07persist.cssUser:Dirkvs/Stylesheets/IC07persist.css2007-10-26T22:19:27Z<p>Dirkvs: </p>
<hr />
<div>#tabs {<br />
/* float:right; */<br />
width:100%;<br />
background:#ffffff;<br />
font-size:100%;<br />
font-weight:bold;<br />
line-height:normal;<br />
border-bottom:1px solid #0a5b86; <br />
}<br />
<br />
#tabs ul {<br />
margin:0;<br />
padding:10px 10px 0 10px;<br />
list-style:none;<br />
}<br />
<br />
#tabs li {<br />
display:inline;<br />
margin:0;<br />
padding:0;<br />
}<br />
<br />
#tabs a {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/06/IC07_tableft.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/8/87/IC07_tabright.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current {<br />
float:left;<br />
background:url("https://static.igem.org/mediawiki/2007/0/03/IC07_tableftactive.png") no-repeat left top;<br />
margin:0;<br />
padding:0 0 0 4px;<br />
text-decoration:none;<br />
}<br />
<br />
#tabs a.current span {<br />
float:left;<br />
display:block;<br />
background:url("https://static.igem.org/mediawiki/2007/4/4d/IC07_tabrightactive.png") no-repeat right top;<br />
padding:5px 15px 4px 6px;<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a:hover {<br />
background-position:0% -42px;<br />
}<br />
<br />
#tabs a:hover span {<br />
background-position:100% -42px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
color:#0a5b86;<br />
}<br />
<br />
#tabs a.current:hover {<br />
background-position:0% 0px;<br />
}<br />
<br />
#tabs a.current:hover span {<br />
background-position:100% 0px;<br />
}</div>Dirkvs