Team:UC Davis/Part1

 kjaBD K            <img alt="" src="http://2009.igem.org/wiki/images/d/d1/UCDAVIS_PIC4.png" style="border: 0px solid ; width: 82px; height: 36px;">   </b></a><b style="color: rgb(255, 255, 153);"><span style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </b><b style="color: rgb(255, 255, 153);"><span style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </b><b style="color: rgb(255, 255, 153);"><span style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">   <img alt="" src="http://2009.igem.org/wiki/images/b/b9/UCDAVIS_PIC8.png" style="border: 0px solid ; width: 78px; height: 36px;"></a>   </b><b style="color: rgb(255, 255, 153);"><span style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">   <img alt="" src="http://2009.igem.org/wiki/images/2/2f/UCDAVIS_PIC5.png" style="border: 0px solid ; width: 81px; height: 36px;"></a>   </b><b style="color: rgb(255, 255, 153);"><span style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </b><b style="color: rgb(255, 255, 153);"><span style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">   <img alt="" src="http://2009.igem.org/wiki/images/a/a6/UCDAVIS_PIC6.png" style="border: 0px solid ; width: 78px; height: 37px;"></a> <img alt="" src="http://2009.igem.org/wiki/images/1/1d/UCDAVIS_PIC7.png" style="border: 0px solid ; width: 83px; height: 37px;"></a>   </b><b style="color: rgb(0, 0, 0);"><span style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </b> <hr style="width: 100%; height: 2px;"> <b style="color: rgb(0, 0, 0); text-decoration: underline;">Parts:</b> <b style="color: rgb(0, 0, 0);"> Parts related to secretion: Parts related to pH sensor: </b> <b style="color: rgb(0, 0, 0);"> </b> <hr style="width: 100%; height: 2px;"> <p class="MsoNormal" style=""><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></a>INPNC </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: Ice-nucleation protein (INP) from Pseudomonas Syringae was suggested to be used for display of foreign proteins on the surface of E.coli(7).Furthermore, researches have shown that an INP derivative constituting the N-and C-terminal domains can and has been used to display foreign proteins on the surface of E.coli(9). <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <p class="MsoNormal" style=""><i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note: A study has shown that "Ice- nucleation Protein (INP), an outer membrane protein from Pseudomonas syringae, is able to catalyze the ice crystal formation of supercooled water. </i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">In our project we are intending to harness and make use of this feature by fusing a specific protein to it. <i>We have modified this protein to Biobrick standard, Tom Knights Standard.</i> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p>  <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%">  <p class="MsoNormal" style=""></a><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: OmpA is one of the proteins on the outer membrane of E.coli (13). OmpA has been found to be useful as utilizable fusion part that can fuse our protein to and display on the surface of E.coli. This part has already been documented on the parts registry; however, it has not been tested via fusion with a target protein linked with a cleavable signal sequence.<u1:p></u1:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <i><span 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. </i><span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p><o:p></o:p> <p class="MsoNormal" style=""><i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note: </i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> “<i>It has remained essentially unknown how proteins of Escherichia coli outer membrane are sorted and incorporated into this membrane” (10) <u1:p></u1:p>For more information go to:</i> <i><span style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836 </i></a> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""></a><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>RBS </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: Ribosome Binding site number 32 (BBa_J61132) from the registry is being used in our secretion system.<u1:p></u1:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For more information go to: </i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <i><span style="">http://partsregistry.org/wiki/index.php/Part:BBa_J61132 </i></a> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""></a><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>Terminator </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: We are using BBa_B0015, a double terminator, as our terminator in both our secretion and pH system.<u1:p></u1:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For more information go to: </i><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <i><span style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 </i></a> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>GFP </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: We are using Green Fluorescent Protein as a reporter that also serves as a small protein in testing our secretion system. <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="Luciferase"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>Luciferase </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: Luciferase is a firefly protein that also fluoresces, so it serves as a reporter as well as a testable large protein.<i> More can be found in:</i> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="LacI"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">LacI </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: One inducible Promoter which was found in the part registry.<i> More can be found in: </i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010"><i><span style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 </i></a> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal"><a name="SS"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: This signal sequence, when placed between INPNC, contains a cleavable site that allows the target fusion protein to ‘secrete’ from INPNC. We will do the same with OmpA. <u1:p></u1:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <p class="MsoNormal" style=""><i><span 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. </i><span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="His"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">6-His Tag </b><span style="font-size: 13pt; line-height: 115%; 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.<u1:p></u1:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <i><span style="font-size: 13pt; line-height: 115%; 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. </i><span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="ChvI_promoter"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">ChvI promoter </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: Gene fusion studies confirmed that ChvI gene was induced by acidic conditions (1). Also, it has been known to implicate in virulence (1). This gene is one of the candidates to be use in our biological pH sensor as a promoter. <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a name="katA"></a>KatA promoter </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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Chromosomal gene is located on the linear chromosome (2) and it seems to be induced under an acidic environment as well as being involved in the <i>Agrobacterium tumorigenesis</i> (2).Research has suggested that ChvG is needed for "responsiveness of gene expression to low pH "(2). This gene has become a candidate to complete our pH sensor device from this evidence. <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="aopB"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">AopB promoter </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: This Chromosomal gene located on the circular chromosome (2) encodes an outer member protein exposed on the bacterial cell surface (2). Also, ChvG was shown to be absolutely required for this gene expression (2)It seems to get induced under an acidic environment as well as being involved in the Agrobacterium tumorigenesis (2). Therefore, we have chosen this gene to be one of our candidates to complete our pH sensor device. <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="PhoA"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>PhoA promoter </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: There has been a suggestion that ChvI can activate AP activity by activating transcription of this gene, PhoA (3). Therefore, this gene has become one of our candidates to complete our pH sensor device. <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <hr align="center" size="2" width="100%"> <p class="MsoNormal"><a name="impA"></a><b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><u1:p></u1:p>ImpA promoter: </b><span style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Gene fusion studies confirmed that impA genes was induced by acidic conditions (1), therefore, this is one of our candidates to complete our pH sensor device. <b> <span style="font-size: 12pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> </b> <p class="MsoNormal"><span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p> </o:p> <hr style="width: 100%; height: 2px;"><small style="font-weight: bold;"><span style="font-size: 18pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">