Team:HKU HongKong/Project/Background.html

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       <p>&nbsp;</p>
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<li class="topfirst"><a href="https://2012.igem.org/Team:HKU_HongKong" style="height:26px;line-height:26px;">
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<li class="topfirst"><a href="https://2012.igem.org/Team:HKU_HongKong" style="height:26px;line-height:26px;">&nbsp;&nbsp;Home&nbsp;&nbsp;</a></li>
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<font color="#232323">&nbsp;&nbsp;Home&nbsp;&nbsp;</font></a></li>
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<li class="topmenu"><a href="#" style="height:26px;line-height:26px;">&nbsp;&nbsp;Team&nbsp;&nbsp;</a>
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<font color="#232323">&nbsp;&nbsp;Team&nbsp;&nbsp;</font></a><font color="#232323">
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Team/About_Us.html">
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Team/About_Us.html">About Us</a></li></font>
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<font color="#232323">About Us</font></a><font color="#232323"></li></font></font><font color="#232323">
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Team/Profiles.html">Profiles</a></li></font>
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Team/Profiles.html">
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<font color="#232323">Profiles</font></a></li></font>
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     <li class="topmenu"><a href="#" style="height:26px;line-height:26px;">&nbsp;&nbsp;Project&nbsp;&nbsp;</a>
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<font color="#232323">&nbsp;&nbsp;Project&nbsp;&nbsp;</font></a><font color="#232323">
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     <ul>
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Project/Background.html">
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Project/Background.html">Background</a></li></font>
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<font color="#232323">Background</font></a><font color="#232323"></li></font></font><font color="#232323">
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Project/Future_Implications.html">Future Implications</a></li></font>
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Project/Future_Implications.html">
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<font color="#232323">Future Implications</font></a></li></font>
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     <li class="topmenu"><a href="#" style="height:26px;line-height:26px;">&nbsp;&nbsp;Data&nbsp;&nbsp;</a>
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<font color="#232323">&nbsp;&nbsp;Data&nbsp;&nbsp;</font></a><font color="#232323">
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     <ul>
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     <li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Data/Protocols.html">
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     <li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Data/Protocols.html">Protocols</a></li></font>
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<font color="#232323">Protocols</font></a><font color="#232323"></li></font></font><font color="#232323">
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Data/Weekly_Notebook.html">Weekly Notebook</a></li></font>
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</font>
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       <li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Data/Bio_Bricks.html">Bio Bricks</a></li></font>
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Data/Weekly_Notebook.html">
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<li><font size="10"><a href="https://2012.igem.org/Team:HKU_HongKong/Data/Results.html">Results</a></li></font>
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<font color="#232323">Weekly Notebook</font></a><font color="#232323"></li></font></font><font color="#232323">
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<font color="#232323">Results</font></a></li></font>
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         </ul>
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<li class="topmenu"><a href="https://2012.igem.org/Team:HKU_HongKong/Safety.html" style="height:26px;line-height:26px;">
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<li class="topmenu"><a href="https://2012.igem.org/Team:HKU_HongKong/Safety.html" style="height:26px;line-height:26px;">&nbsp;&nbsp;Safety&nbsp;&nbsp;</a></li>     
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<font color="#232323">&nbsp;&nbsp;Safety&nbsp;&nbsp;</font></a></li>     
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     <li class="topmenu"><a href="https://2012.igem.org/Team:HKU_HongKong/Attributions.html" style="height:26px;line-height:26px;">&nbsp;&nbsp;Attributions&nbsp;&nbsp;</a></li>
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     <li class="toplast"><a href="https://2012.igem.org/Team:HKU_HongKong/Human_Practise.html" style="height:26px;line-height:26px;">&nbsp;&nbsp;Human Practise&nbsp;&nbsp;</a></li>
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<font color="#232323">&nbsp;&nbsp;Attributions&nbsp;&nbsp;</font></a></li>
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     <li class="toplast"><a href="https://2012.igem.org/Team:HKU_HongKong/Human_Practise.html" style="height:26px;line-height:26px;">
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<font color="#232323">&nbsp;&nbsp;Human Practise&nbsp;&nbsp;</font></a></li>
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</ul>
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<p>&nbsp;</p>
 
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<h2 style="font-variant: normal; vertical-align: baseline; clear: left; color: #232323; font-family: Gentium Basic; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin: 0.7em 0px; padding: 0px">
 
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<span style="font-weight: 400; text-decoration:underline">
 
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<font face="Trebuchet MS" size="6">Abstract</font></span></h2>
 
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<p style="text-align: left; font-style: normal; font-variant: normal; font-weight: normal; font-size: 13px; vertical-align: baseline; color: rgb(85, 85, 85); font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px">
 
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&nbsp;</p>
 
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<p style="text-align: left; font-style: normal; font-variant: normal; font-weight: normal; font-size: 13px; vertical-align: baseline; color: #232323; font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px">
 
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HKU’s iGEM team aims to introduce
 
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an acyl homoerine lactone (AHL)-degrading genetic system into the non-biolfilm-forming
 
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and non-virulent BL21 Escherichia coli strain. PvdQ, an enzyme naturally
 
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produced by Pseudomonas aeruginosa, is an acylase that functions to
 
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degrade long chain AHLs that bacteria like Pseudomonas putida or
 
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aeruginosa itself utilize for biofilm formation. Biofilms are population
 
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density-dependent structures formed by quorum sensing bacteria that
 
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produce and secrete auto-inducers, which signal selective gene
 
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transcription. These signaling molecules, namely the AHLs, are
 
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responsible for most bacterial pathogenicity including the opportunistic
 
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respiratory infections caused by P.aeuroginosa in immunocompromised
 
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patients. </p>
 
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<p style="text-align: left; font-style: normal; font-variant: normal; font-weight: normal; font-size: 13px; vertical-align: baseline; color: #232323; font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px">
 
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As a step towards combating these infections, E.coli can be effectively
 
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used as a protein factory to maximize pvdQ yield in vitro or ex vivo.
 
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Our most preliminary biobrick is a constitutive promoter that drives
 
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baseline, exponential expression of pvdQ. This genetic pathway is
 
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advantageous because the pvdQ gene is constitutively transcribed
 
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regardless of environmental and endogenous factors.&nbsp; </p>
 
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<p style="text-align: left; font-style: normal; font-variant: normal; font-weight: normal; font-size: 13px; vertical-align: baseline; color: rgb(85, 85, 85); font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px">
 
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<font color="#232323">&nbsp;This synthetic genetic pathway is an auto-inductive system where pvdQ
 
-
protein production will specifically depend on the presence of N-dodecanoyl-L-Homoserine
 
-
lactone and its coupling to the LuxR protein. Furthermore, several
 
-
derivatives of the genetic system design can desirably optimize pvdQ
 
-
yield. For instance, implementation of a positive feedback loop will
 
-
upregulate luxR production by the simple placement of the luxR gene
 
-
downstream of PluxR Larger amounts of luxR will therefore bind a greater
 
-
number of AHL molecules secreted by P.aeuroginosa biofilms, thereby
 
-
activating the acylase gene’s expression at a low cell density. Hence,
 
-
the final biobrick produced by iGEM HKU is an AHL-inducible acylase
 
-
system. </font> </p>
 
-
<p style="text-align: left; font-style: normal; font-variant: normal; font-weight: normal; font-size: 13px; vertical-align: baseline; color: #232323; font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px">
 
-
&nbsp;Although the synthetic E.coli cannot be introduced into infected humans
 
-
or soil and water (sources of P.aueroginosa) itself, it can be used to
 
-
mass-produce pvdQ which can then be packaged into small protein-delivery
 
-
bores. These structures can be stimulated to efficiently release pvdQ at
 
-
the desired location, mimicking conventional drug-delivery systems.
 
-
While the mechanism of pvdQ delivery will not be addressed, it can be
 
-
regarded as a potential implication of HKU’s iGEM project.
 
-
 
-
<p style="text-align: left; font-style: normal; font-variant: normal; font-weight: normal; vertical-align: baseline; color: rgb(85, 85, 85); font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px">
 
-
&nbsp;<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
 
-
<u><font face="Trebuchet MS">
 
-
<b><font size="6" color="#232323">M</font></b><font size="6" color="#232323">aterials
 
-
&amp; </font></font><font face="Trebuchet MS" size="6" color="#232323">
 
-
Methods</font></u></p>
 
-
<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
 
-
<i>
 
-
<font color="#232323">Cloning and expressing pvdQ in E. coli</font></i></p>
 
-
<p style="text-align: left; font-style: normal; font-variant: normal; font-weight: normal; font-size: 13px; vertical-align: baseline; color: #232323; font-family: Lato, Tahoma, Arial, sans-serif; letter-spacing: normal; line-height: 19.5px; orphans: 2; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-size-adjust: auto; -webkit-text-stroke-width: 0px; border: 0px none; margin-left: 0px; margin-right: 0px; margin-top: 0px; margin-bottom: 20px; padding: 0px">pvdQ was amplified from genomic DNA of Pseudomonas
 
-
Aeruginosa. A functional biobrick is constructed by combining pvdQ and
 
-
some regulatory elements (such as promoter and terminator). The
 
-
regulatory components are obtained from the iGEM Distribution Kit 2012.
 
-
As a result, a variety of pvdQ regulatory systems can be established.
 
-
Among the various regulation systems, the luxR regulation system is most
 
-
concerned. luxR is a gene that can encode LuxR which binds with AHLs and
 
-
upregulates the luxRp. As a result, in our biobrick model, the
 
-
expression of pvdQ will be upregulated. Increase in production of pvdQ
 
-
indicates an increase in acylase activity, which further degrades AHLs.
 
-
The product biobrick will be a AHL-inducible acylase system. PvdQ will
 
-
only be produced w</font></font><font size="2" color="#232323">hen AHL is present.</font></font></p>
 
-
<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
 
-
<font color="#232323"><i>Testing the inhibitory effect</i></font></p>
 
-
<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
 
-
<font color="#232323" face="Tahoma" size="2">The growth rate of monospecies biofilm of <i>
 
-
Pseudomonas putida</i> is used to reflect the inhibitory effect of
 
-
engineered <i>Escherichia coli</i>. This is because the major AHLs
 
-
secreted by it involve 3-oxo-C12, which is a AHL that can be degraded by
 
-
PvdQ and is also the major AHL produced in Pseudomonas areuginosa, the
 
-
pathogenic microorganism. <i>pvdQ</i> expressing E. coli will be mixed
 
-
with Pseudomonas putida and grown on agar plate. The reduction in
 
-
biofilm formation will be assayed by crystal violet assay. The next part
 
-
of the experiment is to add engineered E. coli to different
 
-
phases of biofilm to validate the role of AHLs in biofilm formation.</font></p>
 
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Revision as of 08:20, 23 September 2012

Team:HKU Hong Kong - 2012

Team:HKU HK

From 2011.igem.org