Team:UC Davis/Adding secretion/model 5

From 2009.igem.org

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<title>Model 1</title>
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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="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"><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_Us"><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="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/Project"><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/Notebook"><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/Parts"><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 5:<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;"><b><span
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
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name="INPNC"></a>INPNC:</span></b><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Ice-nucleation
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protein (INP) from Pseudomonas
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Syringae was suggested to be used for display of foreign proteins on
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the
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surface of <i>E. coli</i>(7).Furthermore, researches have shown that
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an INP
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derivative constituting the N-and C-terminal domains can and has been
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used to
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display foreign proteins on the surface of <i>E. coli</i>(9). In our
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project we
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are intending to harness and make use of this feature by fusing a
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specific
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protein to it. <o:p></o:p></span><br>
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<p class="MsoNormal" style="line-height: normal;"><i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
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have
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modified this protein to Biobrick standard, Tom Knights Standard.<br>
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For more information go to: <a
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href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008">BBa_K265008</a>&nbsp;</span></i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span></p>
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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align="center"><span
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style="font-size: 12pt; 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="line-height: normal;"><a name="OmpA"></a><b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA</span></b><span
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style="font-size: 18pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">&nbsp;</span><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
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is one of the proteins on the outer
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membrane of <i>E. coli</i> (13). OmpA has been found to be useful as
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utilizable
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fusion part that can fuse our protein to and display on the surface of <i>E.
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coli</i>. This part has already been documented on the parts registry;
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however,
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it has not been tested via fusion with a target protein linked with a
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cleavable
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signal sequence. <o:p></o:p></span></p>
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<p class="MsoNormal" style="line-height: normal;"><i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
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have
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modified this protein to Biobrick standard, Tom Knights Standard.</span></i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span><br>
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<i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
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“It has
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remained essentially unknown how proteins of E. coli outer membrane are
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sorted
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and incorporated into this membrane” (10)</span></i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span><br>
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<i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
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more
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information go to:<a
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href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006">
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BBa_K103006</a></span></i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <a
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href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836"><i><span
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style="color: blue;"></span></i></a><o:p></o:p></span></p>
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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align="center"><span
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style="font-size: 12pt; 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="line-height: normal;"><a name="RBS"></a><b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">RBS</span></b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:&nbsp;
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</span><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Ribosome
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Binding site number 32 (BBa_J61132)
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from the registry is being used in our secretion system. <o:p></o:p></span><br>
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<i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
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more
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information go to:</span></i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <big><a
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href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><small><span
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style="font-style: italic;">BBa_J61132</span></small></a></big><a
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href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i><span
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style="color: blue;"></span></i></a><o:p></o:p></span></p>
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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align="center"><span
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style="font-size: 12pt; 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="line-height: normal;"><a name="Terminator"></a><b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Terminator</span></b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
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are using BBa_B0015, a double terminator, as
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our terminator in both our secretion and pH system.<o:p></o:p></span><br>
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<i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
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more
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information go to:</span></i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <a
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href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><span
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style="font-style: italic;">BBa_B0015</span></a> <a
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href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i><span
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style="color: blue;"></span></i></a><o:p></o:p></span></p>
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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align="center"><span
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style="font-size: 12pt; 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="line-height: normal;"><a name="GFP"></a><b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">GFP</span></b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
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are using Green Fluorescent Protein as a
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reporter that also serves as a small protein in testing our secretion
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system.<br>
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<span style="font-style: italic;">For more informaiton go to: </span><a
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style="font-style: italic;"
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href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003">BBa_K265003</a><span
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style="font-style: italic;">&nbsp;</span><o:p></o:p></span></p>
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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align="center"><span
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style="font-size: 12pt; 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="line-height: normal;"><a name="Luciferase"></a><b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Luciferase</span></b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Luciferase
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is a firefly protein that also
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fluoresces, so it serves as a reporter as well as a testable large
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protein.<br>
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<span style="font-style: italic;">For more information go to: <a
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href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019">BBa_1712019</a></span>
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<o:p></o:p></span></p>
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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align="center"><span
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style="font-size: 12pt; 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="line-height: normal;"><a name="LacI"></a><b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">LacI</span></b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">One
+
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inducible Promoter which was found in the
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part registry.<br>
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</span><i><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i><i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
+
-
more information go to:<a
+
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010">
+
-
BBa_R0010</a><br>
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</span></i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
+
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href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010"><i><span
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style="color: blue;"></span></i></a><o:p></o:p></span></p>
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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align="center"><span
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style="font-size: 12pt; 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="line-height: normal;"><a name="SS"></a><b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS</span></b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">This
+
-
signal sequence, when placed between
+
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INPNC, contains a cleavable site that allows the target fusion protein
+
-
to
+
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‘secrete’ from INPNC. We will do the same with OmpA. <o:p></o:p></span><br>
+
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<i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
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have
+
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modified this protein to Biobrick standard, Tom Knights Standard.<br>
+
-
For more infromation go to :<a
+
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href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002">BBa_K265002</a></span></i><span
+
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span></p>
+
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<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
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align="center"><span
+
-
style="font-size: 12pt; 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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<a name="His"></a><b><span
+
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style="font-size: 13pt; 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; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
+
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">The
+
-
6-Histidine Tag serves as a tag for Western
+
-
Blotting if our fluorescent reporters are not expressed as highly as we
+
-
would
+
-
like. <o:p></o:p></span><br>
+
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<small><i><span
+
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
+
-
We are
+
-
using this tag, just in case if the GFP or Luciferase does not work
+
-
under a
+
-
plate reader.</span></i></small>
+
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<hr style="width: 100%; height: 2px;"><span
+
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span><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>
+

Latest revision as of 16:44, 21 October 2009