Team:UC Davis/Adding secretion/model 5

From 2009.igem.org

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<title>Model 1</title>
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<body>
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<div style="text-align: center;"><span
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style="color: rgb(255, 255, 153);"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><span
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style="font-weight: bold;">=</span></span></span><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><big><big><big><big><a
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href="https://2009.igem.org/Team:UC_Davis/homepage2"><img alt=""
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src="https://static.igem.org/mediawiki/2009/a/a4/UCDAVIS_PIC3.png"
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style="border: 0px solid ; width: 83px; height: 36px;"></a> </big></big></big></big></span></b><a
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href="https://2009.igem.org/Team:UC_Davis/About_Us1"><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><big><big><big><big><img
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alt="" src="https://static.igem.org/mediawiki/2009/d/d1/UCDAVIS_PIC4.png"
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style="border: 0px solid ; width: 82px; height: 36px;"></big></big></big></big></span></b></a><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></b><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></b><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><big><big><big><big>
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</big></big></big></big></span></b><b style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><big><big><big><big><a
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href="https://2009.igem.org/Team:UC_Davis/Project1"><img alt=""
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src="https://static.igem.org/mediawiki/2009/b/b9/UCDAVIS_PIC8.png"
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style="border: 0px solid ; width: 78px; height: 36px;"></a> </big></big></big></big></span></b><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><big><big><big><big><a
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href="https://2009.igem.org/Team:UC_Davis/Notebook1"><img alt=""
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src="https://static.igem.org/mediawiki/2009/2/2f/UCDAVIS_PIC5.png"
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style="border: 0px solid ; width: 81px; height: 36px;"></a> </big></big></big></big></span></b><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></b><b
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style="color: rgb(255, 255, 153);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><big><big><big><big><a
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href="https://2009.igem.org/Team:UC_Davis/Part1"><img alt=""
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src="https://static.igem.org/mediawiki/2009/a/a6/UCDAVIS_PIC6.png"
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style="border: 0px solid ; width: 78px; height: 37px;"></a></big></big></big></big></span></b><b
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style="color: rgb(0, 0, 0);"><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></b></div>
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<hr style="width: 100%; height: 2px;"><big><big><big><span
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style="text-decoration: underline; font-weight: bold;"><small>Secretion
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Model
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1:<br>
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<br>
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</small></span></big></big></big><big><span style="font-style: italic;">Click
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on an
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individual part for more information.<br>
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</span></big>
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<div style="text-align: center;"><big><big><big><span
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style="font-weight: bold;"><small><span
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style="text-decoration: underline; background-color: rgb(255, 255, 0);"><a
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href="#LacI"><img alt=""
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src="https://static.igem.org/mediawiki/2009/4/4e/UCDAVIS_PROMOTER_1.png"
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style="border: 0px solid ; width: 242px; height: 58px; background-color: rgb(255, 255, 255);"></a><a
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href="#RBS"><img alt=""
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src="https://static.igem.org/mediawiki/2009/1/15/UCDAVIS_RBS1.png"
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style="border: 0px solid ; width: 89px; height: 81px; background-color: rgb(255, 255, 255);"></a><a
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href="#SS"><img alt=""
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src="https://static.igem.org/mediawiki/2009/9/9d/UCDAVIS_SS1.png"
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style="border: 0px solid ; width: 94px; height: 69px; background-color: rgb(255, 255, 255);"></a><img
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style="width: 106px; height: 59px; background-color: rgb(255, 255, 255);"
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alt="" src="https://static.igem.org/mediawiki/2009/0/0c/UCDAVIS_GENE1.png"><a
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href="#His"><img alt=""
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src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
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style="border: 0px solid ; width: 134px; height: 66px; background-color: rgb(255, 255, 255);"></a><a
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href="#Terminator"><img alt=""
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src="https://static.igem.org/mediawiki/2009/c/ca/UCDAVIS_STOP.png"
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style="border: 0px solid ; width: 94px; height: 83px; background-color: rgb(255, 255, 255);"></a><br>
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</span></small></span></big></big></big><big><big><big><span
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style="font-weight: bold;"><small><span
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style="text-decoration: underline; background-color: rgb(255, 255, 0);"><br>
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</span>a.<a href="#LacI"><span style="text-decoration: underline;"><img
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alt=""
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src="https://static.igem.org/mediawiki/2009/4/4e/UCDAVIS_PROMOTER_1.png"
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style="border: 0px solid ; width: 200px; height: 52px;"></span></a><span
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style="text-decoration: underline; background-color: rgb(255, 255, 0);"><a
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href="#RBS"><img alt=""
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src="https://static.igem.org/mediawiki/2009/1/15/UCDAVIS_RBS1.png"
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style="border: 0px solid ; width: 69px; height: 63px; background-color: rgb(255, 255, 255);"></a><a
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href="#SS"><img alt=""
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src="https://static.igem.org/mediawiki/2009/9/9d/UCDAVIS_SS1.png"
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style="border: 0px solid ; width: 73px; height: 65px; background-color: rgb(255, 255, 255);"></a><a
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href="#GFP"><img alt=""
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src="https://static.igem.org/mediawiki/2009/7/7d/UCDAVIS_GFP.png"
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style="border: 0px solid ; width: 83px; height: 55px; background-color: rgb(255, 255, 255);"></a><a
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href="#His"><img alt=""
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src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
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style="border: 0px solid ; width: 110px; height: 58px; background-color: rgb(255, 255, 255);"></a><a
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href="#Terminator"><img alt=""
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src="https://static.igem.org/mediawiki/2009/c/ca/UCDAVIS_STOP.png"
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style="border: 0px solid ; width: 78px; height: 71px; background-color: rgb(255, 255, 255);"></a></span></small></span></big></big></big><br>
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<div style="text-align: center;"><big><big><big><span
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style="font-weight: bold;"><small>b.<span
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style="text-decoration: underline;"><a href="#LacI"><img alt=""
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src="https://static.igem.org/mediawiki/2009/4/4e/UCDAVIS_PROMOTER_1.png"
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style="border: 0px solid ; width: 195px; height: 52px;"></a><a
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href="#RBS"><img alt=""
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src="https://static.igem.org/mediawiki/2009/1/15/UCDAVIS_RBS1.png"
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style="border: 0px solid ; width: 68px; height: 63px;"></a><a
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href="#SS"><img alt=""
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src="https://static.igem.org/mediawiki/2009/9/9d/UCDAVIS_SS1.png"
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style="border: 0px solid ; width: 73px; height: 65px;"></a><a
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href="#Luciferase"><img alt=""
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src="https://static.igem.org/mediawiki/2009/9/98/UCDAVIS_Luciferase.png"
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style="border: 0px solid ; width: 87px; height: 49px;"></a><a
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href="#His"><img alt=""
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src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
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style="border: 0px solid ; width: 110px; height: 58px;"></a><a
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href="#Terminator"><img alt=""
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src="https://static.igem.org/mediawiki/2009/c/ca/UCDAVIS_STOP.png"
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style="border: 0px solid ; width: 78px; height: 71px;"></a></span></small></span></big></big></big><big><big><big><span
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style="font-weight: bold;"><small><span
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style="text-decoration: underline; background-color: rgb(255, 255, 0);"></span></small></span></big></big></big></div>
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</div>
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<div style="text-align: center;"><big><span style="font-style: italic;"></span></big><br>
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</div>
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<big><big><big><span
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style="text-decoration: underline; font-weight: bold;"></span></big></big></big>
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<hr style="width: 100%; height: 2px;">
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<p class="MsoNormal"><b><span
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style="font-size: 18pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
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name="INPNC"></a></span></b><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">INPNC</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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Ice-nucleation protein (INP) from Pseudomonas Syringae was
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suggested to be used for display of foreign proteins on the surface of <i>E.coli</i>(7).Furthermore,
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researches have shown that an INP derivative constituting the N-and
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C-terminal
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domains can and has been used to display foreign proteins on the
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surface of <i>E.coli</i>(9).
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</span><span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<p class="MsoNormal" style=""><i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
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A study has shown that "Ice- nucleation Protein (INP), an outer
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membrane
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protein from Pseudomonas syringae, is able to catalyze the ice crystal
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formation of supercooled water.</span></i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">In
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our project we are intending to harness and make use of this feature by
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fusing
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a specific protein to it. <i>We have modified this protein to Biobrick
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standard, Tom Knights Standard.</i></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal" style=""><a name="OmpA"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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OmpA is one of the proteins on the outer membrane of <i>E.coli</i>
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(13). OmpA has been found to be useful as utilizable fusion part that
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can fuse
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our protein to and display on the surface of <i>E.coli</i>. This part
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has
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already been documented on the parts registry; however, it has not been
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tested
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via fusion with a target protein linked with a cleavable signal
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sequence.<u1:p></u1:p></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><br>
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</span><i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
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have modified this protein to Biobrick standard, Tom Knights
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Standard. </span></i><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p><o:p></o:p></span></p>
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<p class="MsoNormal" style=""><i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:</span></i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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“<i>It has
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remained essentially unknown how proteins of Escherichia coli outer
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membrane
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are sorted and incorporated into this membrane” (10)<br>
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<u1:p></u1:p>For more information go to:</i> <a
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href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836"><i><span
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style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836</span></i></a></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal" style=""><a name="RBS"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>RBS</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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Ribosome Binding site number 32 (BBa_J61132) from the registry is
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being used in our secretion system.<u1:p></u1:p></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><br>
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</span><i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
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more information go to:</span></i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i><span
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style="">http://partsregistry.org/wiki/index.php/Part:BBa_J61132</span></i></a></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal" style=""><a name="Terminator"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>Terminator</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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We are using BBa_B0015, a double terminator, as our terminator in
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both our secretion and pH system.<u1:p></u1:p></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><br>
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</span><i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
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more information go to:</span></i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i><span
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style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015</span></i></a></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal" style=""><a name="GFP"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>GFP</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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We are using Green Fluorescent Protein as a reporter that also
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serves as a small protein in testing our secretion system.</span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal" style=""><a name="Luciferase"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>Luciferase</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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Luciferase is a firefly protein that also fluoresces, so it
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serves as a reporter as well as a testable large protein.<i><br>
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More can be found in:</i></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal" style=""><a name="LacI"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">LacI</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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One inducible Promoter which was found in the part registry.<i><br>
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More can be found in: </i><a
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href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010"><i><span
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style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010</span></i></a></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal"><a name="SS"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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This signal sequence, when placed between INPNC, contains a
+
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cleavable site that allows the target fusion protein to ‘secrete’ from
+
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INPNC.&nbsp; We will do the same with OmpA.&nbsp; <u1:p></u1:p></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
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<p class="MsoNormal" style=""><i><span
+
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
-
have modified this protein to Biobrick standard, Tom Knights
+
-
Standard.</span></i><span
+
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
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<hr align="center" size="2" width="100%"></span></div>
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<p class="MsoNormal" style=""><a name="His"></a><b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">6-His
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Tag</span></b><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
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The 6-Histidine Tag serves as a tag for Western Blotting if our
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fluorescent reporters are not expressed as highly as we would like.<u1:p></u1:p></span><span
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><br>
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</span><i><span
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
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We are using this tag, just in case if the GFP or Luciferase
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does not work under a plate reader.</span></i></p>
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<b><i><span
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style="font-size: 12pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i></b><big><big><big><span
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style="text-decoration: underline; font-weight: bold;"></span></big></big></big>
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</body>
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</html>
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Latest revision as of 16:44, 21 October 2009