Team:UC Davis/Parts

 PARTS1            <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);"> <img style="width: 493px; height: 242px;" alt="" src="http://2009.igem.org/wiki/images/5/5e/UCDAVIS_picture1.jpg"></b> <hr style="width: 100%; height: 2px;"> <p class="MsoNormal" style="line-height: normal; font-weight: bold; text-align: center; text-decoration: underline;"> New parts: <span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p> <p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"></a>INPNC: The ice-nucleation protein (INP) from <i>Pseudomonas syringae</i> (9</a>) is used by its natural host to nucleate ice formation and is implicated in P.syringae-associated pathogenesis''. ''INP and a truncated derivative lacking the central domain (INPNC) have been used extensively for displaying proteins on the surface of E. coli (7</a>). For instance, AldO and PhaZ1 have been successfully displayed on the surface of E.coli using INPNC (7, 15</a>). <u1:p></u1:p>Park et al. have shown that when INPNC is fused to the phaZ1 gene and its signal sequence, it can serve as a suitable surface delivery and secretion device of the otherwise toxic phaZ1 gene product(15</a>). <u1:p></u1:p>This part was synthesized by Mr. Gene (Regensburg, Germany) with codon optimization and subsequently transferred into vector (<span style="background: rgb(255, 255, 51) none repeat scroll 0% 50%; color: black; -moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial;"> pSB1AK3</a>). As it is expected that this part will be used in the context of the fusion protein, the prefix and suffix for this part are consistent with the <i>BBF RCF-12</i> standard. <u1:p></u1:p>We have proposed to build and test a general protein secretion system modeled after that developed by Park et al. in which a fusion of INPNC and the signal sequence from the phaZ1 gene are used to secrete any target protein. <u1:p></u1:p><i>We have modified this protein to be consistent with the BBF RFC-12 Standard. We have submitted this part to the parts registry as part </i><i><span style="color: rgb(0, 0, 153);">BBa_K265008 </i></a><i>. </i> <p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"> <u1:p></u1:p><o:p></o:p> <p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"></a><u1:p></u1:p>SS: The signal sequence (SS) for the phaZ1 gene product of <i>Paucimonas lemoignei</i>, a polyhydroxybutyrate depolymerase (15</a>). In the native protein the signal sequence is cleaved between residues Ala37 and Leu38. Park et al. have showed that the fusion of the complete <i>phaZ1 </i>gene (including SS) and a truncated ice nucleation protein from <i>Pseudomonas syringae</i> (<i><span style="color: rgb(0, 0, 153);">BBa_K265008 </i></a>), could lead to stable expression and secretion of the phaZ1 gene product. <u1:p></u1:p>We propose that the signal sequence might be generally useful as a cleavage tag in secretion systems that include a membrane anchor component, such as INPNC (<i><span style="color: rgb(0, 0, 153);">BBa_K265008 </i></a>) or OmpA (<i><span style="color: blue;">BBa_K103006 </a>).<span style="color: rgb(0, 41, 57);"> </i>The proposed constructs would consists of a membrane anchor (INPNC or OmpA) followed by the cleavable signal sequence and finally a target protein marked for secretion. <u1:p></u1:p><i>Since we expect that this part will be used in the context of a fusion protein, we have modified this protein to be consistent with BBF RFC-12 Standard. We have submitted this part to the part registry as part </i><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"><i><span style="color: rgb(0, 0, 153);">BBa_K265002 </i></a>. <u1:p></u1:p> <p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a name="INPNCSS"></a>INPNC+SS: Park et al. have showed that the fusion of the complete phaZ1 gene (including SS) and a truncated ice nucleation protein from Pseudomonas syringae (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span style="color: rgb(0, 0, 153);">BBa_K265008 </i></a>), could lead to stable expression and secretion of the phaZ1 gene product (<a href="http://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>). <u1:p></u1:p>We propose that this system might be generally useful for the secretion of other target proteins in E. coli and have therefore created a fusion of parts <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span style="color: rgb(0, 0, 153);">BBa_K265008 </i></a> (INPNC) and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"><i><span style="color: rgb(0, 0, 153);">BBa_K265002 </i></a> (SS) which is compatible with the ''BBF RFC-12 Standard. <u1:p></u1:p>'' During the construction of this part, two silent mutations were introduced in the coding region of INPNC (T324A and A348T) that differ from those in part <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span style="color: rgb(0, 0, 153);">BBa_K265008 </i></a>. <u1:p></u1:p><i>We have submitted this part to the part registry in the BBF RFC-12 Standard </i>as part <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K265009"><i><span style="color: rgb(0, 0, 153);">BBa_K265009 </i></a>. <u1:p></u1:p> <p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a name="OmpAss"></a>OmpA+SS: Since OmpA is believed to function similarly to INPNC and Park et al. have showed that the fusion of the complete <i>phaZ1 </i>gene (including SS) and a truncated ice nucleation protein from <i>Pseudomonas syringae</i> (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span style="color: rgb(0, 0, 153);">BBa_K265008 </i></a>), could lead to stable expression and secretion of the phaZ1 gene product (<a href="http://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>), we have decided to test and see if OmpA's ability to secret increases when it is used with a signal sequence. <u1:p></u1:p><i>We have modified this protein to be consistent with BBF RFC-12 Standard and have submitted this part to the part registry, </i><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K265011"><i><span style="color: rgb(0, 0, 153);">BBa_K265011 </i> <span style="color: blue;">. </a> <u1:p></u1:p> <hr align="center" size="2" width="100%"> <p class="MsoNormal"><a name="OmpA"></a>OmpA: OmpA is one of the proteins on the outer membrane of E. coli (<a href="http://2009.igem.org/Team:UC_Davis/Contacts_References">13</a>),it is used as a displaying fusion protein on the cell surface. This part has already been documented on the parts registry; however, it has not been tested as a compnent of secretion system (via fusion with a target protein linked with a cleavable signal sequence) <i> <u1:p></u1:p>We have modified this protein to be consistent with BBF RFC-12 Standard. Note: “It has remained essentially unknown how proteins of E. coli outer membrane are sorted to and incorporated into this membrane” (<a href="http://2009.igem.org/Team:UC_Davis/Contacts_References">10</a>)</i> <i> For more information go to:<a href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"> <span style="color: blue;"> BBa_K103006 </a></i> <u1:p></u1:p> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="RBS"></a>RBS: Ribosome Binding site number 32 (BBa_J61132) from the registry is being used in our secretion system. For more information go to: <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i> <span style="color: blue;">BBa_J61132 </i></a> <u1:p></u1:p> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="Terminator"></a>Terminator: We are using BBa_B0015, a double terminator, as our terminator in both our secretion and pH system. For more information go to: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i> <span style="color: blue;">BBa_B0015 </i></a> <u1:p></u1:p> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="GFP"></a>GFP <b>(Green Fluorescent Protein)</b>: Mutant of GFP known to be very stable (superfolder), which will let this protein fold quickly so we can use either a fluorescent reader or UV light to detect it. Therefore it has been used as a reporter in our secretion system. It also serves as a small protein in testing our secretion system. For more informaiton go to: <a href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003"><i> <span style="color: blue;">BBa_K265003 </i></a>  <u1:p></u1:p> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="Luciferase"></a>Luciferase: Luciferase is a firefly protein that also fluoresces, so it serves as a reporter as well as a testable large protein. <i>For more information go to: <a href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019"> <span style="color: blue;">BBa_1712019 </a></i> <u1:p></u1:p> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="LacI"></a>LacI: An inducible promoter that was found in the part registry. <i>For more information go to:<a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010"> <span style="color: blue;"> BBa_R0010 </a> </i> <u1:p></u1:p> <hr align="center" size="2" width="100%"> <p class="MsoNormal" style=""><a name="His"></a>6-His Tag: The 6-Histidine Tag serves as a tag for Western Blotting if our fluorescent reporters are not expressed as highly as we would like. <i>Note: We are using this tag as an additional method for assay beside fluorescence of GFP and Luciferase.</i> <hr align="center" size="2" width="100%"> more information go to: <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&amp;group=UC_Davis"><i> <span style="color: blue;">UCDAVIS_Parts </i></a> <u1:p></u1: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;;">