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<p>&nbsp;</p>
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p>&nbsp;</p>
<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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advantageous because the pvdQ gene is constitutively transcribed  
advantageous because the pvdQ gene is constitutively transcribed  
regardless of environmental and endogenous factors.&nbsp; </p>
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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<br></div>
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<font color="#232323">&nbsp;This synthetic genetic pathway is an auto-inductive system where pvdQ
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protein production will specifically depend on the presence of N-dodecanoyl-L-Homoserine
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<p style="text-align:center;">
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lactone and its coupling to the LuxR protein. Furthermore, several
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derivatives of the genetic system design can desirably optimize pvdQ
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yield. For instance, implementation of a positive feedback loop will
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upregulate luxR production by the simple placement of the luxR gene
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downstream of PluxR Larger amounts of luxR will therefore bind a greater
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number of AHL molecules secreted by P.aeuroginosa biofilms, thereby
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activating the acylase gene’s expression at a low cell density. Hence,
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the final biobrick produced by iGEM HKU is an AHL-inducible acylase
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system. </font> </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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&nbsp;Although the synthetic E.coli cannot be introduced into infected humans
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or soil and water (sources of P.aueroginosa) itself, it can be used to
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mass-produce pvdQ which can then be packaged into small protein-delivery
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bores. These structures can be stimulated to efficiently release pvdQ at
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the desired location, mimicking conventional drug-delivery systems.
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While the mechanism of pvdQ delivery will not be addressed, it can be
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regarded as a potential implication of HKU’s iGEM project.
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<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">
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&nbsp;<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
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<u><font face="Trebuchet MS">
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<b><font size="6" color="#232323">M</font></b><font size="6" color="#232323">aterials
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&amp; </font></font><font face="Trebuchet MS" size="6" color="#232323">
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Methods</font></u></p>
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<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
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<i>
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<font color="#232323">Cloning and expressing pvdQ in E. coli</font></i></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">pvdQ was amplified from genomic DNA of Pseudomonas
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Aeruginosa. A functional biobrick is constructed by combining pvdQ and
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some regulatory elements (such as promoter and terminator). The
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regulatory components are obtained from the iGEM Distribution Kit 2012.
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As a result, a variety of pvdQ regulatory systems can be established.
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Among the various regulation systems, the luxR regulation system is most
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concerned. luxR is a gene that can encode LuxR which binds with AHLs and
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upregulates the luxRp. As a result, in our biobrick model, the
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expression of pvdQ will be upregulated. Increase in production of pvdQ
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indicates an increase in acylase activity, which further degrades AHLs.
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The product biobrick will be a AHL-inducible acylase system. PvdQ will
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only be produced w</font></font><font size="2" color="#232323">hen AHL is present.</font></font></p>
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<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
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<font color="#232323"><i>Testing the inhibitory effect</i></font></p>
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<p class="MsoNormal" style="text-align:justify;text-justify:inter-ideograph">
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<font color="#232323" face="Tahoma" size="2">The growth rate of monospecies biofilm of <i>
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Pseudomonas putida</i> is used to reflect the inhibitory effect of
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engineered <i>Escherichia coli</i>. This is because the major AHLs
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secreted by it involve 3-oxo-C12, which is a AHL that can be degraded by
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PvdQ and is also the major AHL produced in Pseudomonas areuginosa, the
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pathogenic microorganism. <i>pvdQ</i> expressing E. coli will be mixed
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with Pseudomonas putida and grown on agar plate. The reduction in
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biofilm formation will be assayed by crystal violet assay. The next part
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of the experiment is to add engineered E. coli to different
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phases of biofilm to validate the role of AHLs in biofilm formation.</font></p>
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