Team:UC Davis/Parts

From 2009.igem.org

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<title>kjaBD K</title>
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<title>PARTS1</title>
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<div style="text-align: left;"><big><big><b
<div style="text-align: left;"><big><big><b
style="color: rgb(0, 0, 0); text-decoration: underline;">Parts:</b></big></big><b
style="color: rgb(0, 0, 0); text-decoration: underline;">Parts:</b></big></big><b
-
style="color: rgb(0, 0, 0);">&nbsp;&nbsp; <br>
+
style="color: rgb(0, 0, 0);">&nbsp;&nbsp;&nbsp; <br>
-
<br>
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Parts related to
Parts related to
-
secretion:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+
secretion:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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Parts related to pH sensor:<br>
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&nbsp; &nbsp; &nbsp;&nbsp;&nbsp; Parts related to pH
 +
sensor:<br>
</b>
</b>
<table
<table
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<td style="vertical-align: top;">Others:<br>
<td style="vertical-align: top;">Others:<br>
</td>
</td>
-
<td style="vertical-align: top;">Proteins:<br>
+
<td style="vertical-align: top;">&nbsp;New parts:<br>
</td>
</td>
<td style="vertical-align: top;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
<td style="vertical-align: top;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
Promoters:<br>
Promoters:<br>
</td>
</td>
 +
<td style="vertical-align: top;">Proteins:</td>
</tr>
</tr>
<tr>
<tr>
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<td style="vertical-align: top;">
<td style="vertical-align: top;">
<ul>
<ul>
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<li>ChvI</li>
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<li><a href="#INPNCSS">INPNC + SS<br>
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<li>ChvG</li>
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</a></li>
 +
<li><a href="#OmpAss">OmpA + SS<br>
 +
</a></li>
 +
<li><a href="#INPNC">INPNC </a><br>
 +
</li>
 +
<li><a href="#SS">SS</a><br>
 +
</li>
</ul>
</ul>
</td>
</td>
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&nbsp;&nbsp; </li>
&nbsp;&nbsp; </li>
<li>I<a href="#impA">mpA promoter</a></li>
<li>I<a href="#impA">mpA promoter</a></li>
 +
</ul>
 +
</td>
 +
<td style="vertical-align: top;">
 +
<ul>
 +
<li><a href="https://2009.igem.org/Team:UC_Davis/ChvI1">ChvI</a></li>
 +
<li><a
 +
href="https://2009.igem.org/Team:UC_Davis/Project1/ChvG.html">ChvG</a></li>
</ul>
</ul>
</td>
</td>
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</tbody>
</tbody>
</table>
</table>
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<b style="color: rgb(0, 0, 0);">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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<div style="text-align: center;"><b style="color: rgb(0, 0, 0);">&nbsp;
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</b><br>
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<img style="width: 493px; height: 242px;" alt=""
 +
src="https://static.igem.org/mediawiki/2009/5/5e/UCDAVIS_picture1.jpg"></b><br>
 +
</div>
<hr style="width: 100%; height: 2px;">
<hr style="width: 100%; height: 2px;">
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<p><a name="INPNC"></a><big><span style="font-weight: bold;">INPNC:</span></big>Ice-nucleation
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<p class="MsoNormal"
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protein (INP) from Pseudomonas Syringae was suggested
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style="line-height: normal; font-weight: bold; text-align: center; text-decoration: underline;"><big><big>New
-
to be used for display of foreign proteins on the surface of <i>E. coli</i>(7).Furthermore,
+
parts:</big></big>&nbsp;</p>
-
researches have shown that an INP derivative constituting the N-and
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<span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span>
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C-terminal domains can and has been used to display foreign proteins on
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<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
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the surface of <i>E. coli</i>(9). In our project we are intending to
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name="INPNC"></a><b>INPNC:</b> The ice-nucleation protein (INP) from <i>Pseudomonas
-
harness and make use of this feature by fusing a specific protein to
+
syringae</i> (<a
-
it. </p>
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">9</a>)
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<p><span style="font-style: italic;">We have modified this protein to
+
is used by its natural host to nucleate ice formation and
-
Biobrick standard, Tom Knights Standard.</span> </p>
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is implicated in<i> P.syringae</i>-associated pathogenesis<i>.&nbsp; </i>INP
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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and
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style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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a truncated derivative lacking the central domain (INPNC) have been
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<hr align="center" size="2" width="100%"></span></div>
+
used
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<p><a name="OmpA"></a><big><b><span
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extensively for displaying proteins on the surface of <i>E. coli </i>(<a
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA</span></b></big><span
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">7</a>).&nbsp;
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><big>:</big>&nbsp;</span><span
+
For instance, AldO and PhaZ1 have been successfully displayed on the
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style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i></i></span>OmpA
+
surface of
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is one of the proteins on the outer membrane of <i>E. coli</i> (13).
+
<i>E.coli </i>using INPNC (<a
-
OmpA has been found to be useful as utilizable fusion part that can
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">7, 15</a>).
-
fuse our protein to and display on the surface of <i>E. coli</i>.
+
<br>
-
This part has already been documented on the parts registry; however,
+
<u1:p></u1:p>Park <i>et al.</i> have shown that when INPNC is fused to
-
it has not been tested via fusion with a target protein linked with a
+
the <i>phaZ1</i>
-
cleavable signal sequence.
+
gene and its signal sequence, it can serve as a suitable surface
 +
delivery
 +
and secretion device of the otherwise toxic <i>phaZ1</i> gene product(<a
 +
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).&nbsp;
 +
<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;"></span>
 +
<a href="http://partsregistry.org/Part:pSB1AK3">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.&nbsp; <br>
 +
<u1:p></u1:p>We have proposed to build and test a general protein
 +
secretion
 +
system modeled after that developed by Park <i>et al. </i>in which a
 +
fusion of
 +
INPNC and the signal sequence from the <i>phaZ1</i> gene are used to
 +
secrete
 +
any target protein.&nbsp; <br>
 +
<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><a
 +
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span
 +
style="color: rgb(0, 0, 153);">BBa_K265008</span></i></a><i>.<br>
 +
</i></p>
 +
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><br>
 +
<i><u1:p></u1:p></i><o:p></o:p></p>
 +
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
 +
name="SS"></a><b><u1:p></u1:p>SS:</b> The
 +
signal sequence (SS) for the <i>phaZ1 </i>gene product of <i>Paucimonas
 +
lemoignei</i>, a polyhydroxybutyrate depolymerase (<a
 +
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).&nbsp;
 +
In the
 +
native
 +
protein the signal sequence is cleaved between residues Ala37 and
 +
Leu38.&nbsp;
 +
Park <i>et al. </i>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</span></i></a>), could lead
 +
to stable
 +
expression and secretion of the <i>phaZ1</i> gene product.&nbsp; <br>
 +
<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 (<a
 +
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span
 +
style="color: rgb(0, 0, 153);">BBa_K265008</span></i></a>) or OmpA (<i><a
 +
href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"><span
 +
style="color: blue;">BBa_K103006</span></a>).<span
 +
style="color: rgb(0, 41, 57);"> </span></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.&nbsp; <br>
 +
<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</span></i></a>.<br>
 +
<br>
</p>
</p>
-
<p><span style="font-style: italic;">We have modified this protein to
+
<u1:p></u1:p>
-
Biobrick standard, Tom Knights Standard.</span> </p>
+
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
-
<p><i>Note: “It has remained essentially unknown how proteins of E.
+
name="INPNCSS"></a><b>INPNC+SS:</b> Park <i>et al. </i>have showed
-
coli outer membrane are sorted and incorporated into this membrane” (10)</i>
+
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</span></i></a>), could lead
 +
to stable
 +
expression and secretion of the <i>phaZ1</i> gene product (<a
 +
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).<br>
 +
<u1:p></u1:p>We propose that this system might be generally useful for
 +
the
 +
secretion of other target proteins in <i>E. coli</i> 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</span></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</span></i></a> (SS) which
 +
is compatible with
 +
the <i>BBF RFC-12 Standard. <u1:p></u1:p></i><br>
 +
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</span></i></a>.&nbsp; <br>
 +
<u1:p></u1:p><i>We have submitted this part to the part registry in the
 +
BBF
 +
RFC-12 Standard </i>as part<i> </i><a
 +
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K265009"><i><span
 +
style="color: rgb(0, 0, 153);">BBa_K265009</span></i></a>.<br>
 +
<br>
</p>
</p>
-
<p class="MsoNormal" style=""><span
+
<u1:p></u1:p>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i><u1:p></u1:p><small>For
+
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
-
more information go to:</small></i><small> <a
+
name="OmpAss"></a><b>OmpA+SS:</b> Since OmpA is believed to function
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836"><i><span
+
similarly
-
style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836</span></i></a></small></span><span
+
to INPNC and Park <i>et al. </i>have showed that the fusion of the
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
complete <i>phaZ1
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
</i>gene (including SS) and a truncated ice nucleation protein from <i>Pseudomonas
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
syringae</i> (<a
-
<hr align="center" size="2" width="100%"></span></div>
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span
-
<p class="MsoNormal" style=""><a name="RBS"></a><b><span
+
style="color: rgb(0, 0, 153);">BBa_K265008</span></i></a>), could lead
-
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
+
to stable
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:&nbsp;
+
expression and secretion of the <i>phaZ1</i> gene product (<a
-
</span>Ribosome Binding site number 32 (BBa_J61132) from the registry
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>),
-
is being used in our secretion system. <span
+
we
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><br>
+
have decided
-
</span>
+
to test and see if OmpA's ability to secret increases when it is used
-
</p>
+
with a
-
<p class="MsoNormal"><i>For more information go to:</i> <a
+
signal sequence.<br>
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i>http://partsregistry.org/wiki/index.php/Part:BBa_J61132</i></a></p>
+
<u1:p></u1:p><i>We have modified this protein to be consistent with BBF
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
RFC-12
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
Standard and have submitted this part to the part registry, </i><a
-
<hr align="center" size="2" width="100%"></span></div>
+
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K265011"><i><span
-
<p class="MsoNormal" style=""><a name="Terminator"></a><b><span
+
style="color: rgb(0, 0, 153);">BBa_K265011</span></i><u><span
-
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
+
style="color: blue;">.</span></u></a></p>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
+
<u1:p></u1:p>
-
</span>We are using BBa_B0015, a double terminator, as our terminator
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
in both our secretion and pH system.<br>
+
<hr align="center" size="2" width="100%"></div>
-
</p>
+
<p class="MsoNormal"><a name="OmpA"></a><b>OmpA</b>: OmpA is one of the
-
<p class="MsoNormal"><i>For more information go to:</i> <a
+
proteins on
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i>http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015</i></a></p>
+
the outer membrane of <i>E. coli</i> (<a
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">13</a>),it
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
is used as a displaying
-
<hr align="center" size="2" width="100%"></span></div>
+
fusion
-
<p class="MsoNormal" style=""><a name="GFP"></a><b><span
+
protein on the cell surface . This part has already been documented on
-
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
+
the parts
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
+
registry; however, it has not been tested as a compnent of secretion
-
</span>We are using Green Fluorescent Protein as a reporter that also
+
system
-
serves as a small protein in testing our secretion system.<span
+
(via fusion with a target protein linked with a cleavable signal
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
sequence) <i><br>
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
<u1:p></u1:p>We have modified this protein to be consistent with BBF
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
RFC-12
-
<hr align="center" size="2" width="100%"></span></div>
+
Standard.<br>
-
<p class="MsoNormal" style=""><a name="Luciferase"></a><b><span
+
Note: “It has remained essentially unknown how proteins of E. coli
-
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
+
outer
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
+
membrane are sorted to and incorporated into this membrane” (<a
-
</span>Luciferase is a firefly protein that also fluoresces, so it
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">10</a>)</i>
-
serves as a reporter as well as a testable large protein.
+
<i><br>
-
<span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
For more information go to:<a
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"><u><span
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
style="color: blue;"> BBa_K103006</span></u></a></i> </p>
-
<hr align="center" size="2" width="100%"></span></div>
+
<u1:p></u1:p>
-
<p class="MsoNormal" style=""><a name="LacI"></a><b><span
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">LacI</span></b><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
+
<p class="MsoNormal" style=""><a name="RBS"></a><b>RBS</b>:&nbsp;
-
</span>One inducible Promoter which was found in the part registry.<br>
+
Ribosome Binding site number 32 (BBa_J61132)
-
<small><span
+
from the registry is being used in our secretion system. <br>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i><br>
+
<i>For more information go to:</i> <a
-
<small>More can be found in: </small></i><small><a
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i><u><span
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010"><i><span
+
style="color: blue;">BBa_J61132</span></u></i></a></p>
-
style="">http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010</span></i></a></small></span></small><span
+
<u1:p></u1:p>
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<p class="MsoNormal" style=""><a name="Terminator"></a><b>Terminator</b>:
-
<hr align="center" size="2" width="100%"></span></div>
+
We are using BBa_B0015, a double
-
<p><a name="SS"></a><b><span
+
terminator, as our terminator in both our secretion and pH system.<br>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS</span></b><span
+
<i>For more information go to:</i> <a
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span>This
+
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i><u><span
-
signal sequence, when placed between INPNC, contains a
+
style="color: blue;">BBa_B0015</span></u></i></a> </p>
-
cleavable site that allows the target fusion protein to ‘secrete’ from
+
<u1:p></u1:p>
-
INPNC. We will do the same with OmpA. </p>
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
<p><span style="font-style: italic;">We have modified this protein to
+
<hr align="center" size="2" width="100%"></div>
-
Biobrick standard, Tom Knights Standard.</span> </p>
+
<p class="MsoNormal" style=""><a name="GFP"></a><b>GFP</b> <b>(Green
-
<p class="MsoNormal" style=""><span
+
Fluorescent Protein)</b>: Mutant of GFP
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
known to be very stable (superfolder), which will let this protein fold
-
</span><i><span
+
quickly
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i><span
+
so we can use either a fluorescent reader or UV light to detect it.
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
Therefore
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
it has been used as a reporter in our secretion system. It also serves
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
as a
-
<hr align="center" size="2" width="100%"></span></div>
+
small protein in testing our secretion system.<br>
-
<p><a name="His"></a><b><span
+
<i>For more informaiton go to: </i><a
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">6-His
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003"><i><u><span
-
Tag</span></b><span
+
style="color: blue;">BBa_K265003</span></u></i></a><i>&nbsp;</i></p>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span>The
+
<u1:p></u1:p>
-
6-Histidine Tag serves as a tag for Western Blotting if our
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
fluorescent reporters are not expressed as highly as we would like.
+
<hr align="center" size="2" width="100%"></div>
-
</p>
+
<p class="MsoNormal" style=""><a name="Luciferase"></a><b>Luciferase</b>:
-
<p><i>Note: We are using this tag, just in case if the GFP or
+
Luciferase is a firefly protein that
-
Luciferase does not work under a plate reader.</i>
+
also fluoresces, so it serves as a reporter as well as a testable large
-
</p>
+
protein.<br>
-
<p class="MsoNormal" style=""><i><span
+
<i>For more information go to: <a
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019"><u><span
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
style="color: blue;">BBa_1712019</span></u></a></i> </p>
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
<u1:p></u1:p>
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
<hr align="center" size="2" width="100%"></span></div>
+
<hr align="center" size="2" width="100%"></div>
-
<p class="MsoNormal" style=""><a name="ChvI_promoter"></a><b><span
+
<p class="MsoNormal" style=""><a name="LacI"></a><b>LacI</b>: An
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">ChvI
+
inducible promoter that was found in the part
-
promoter</span></b><span
+
registry.<br>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
+
<i>For more information go to:<a
-
</span><small><span style="font-size: 13pt; line-height: 115%;"><small>Gene
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010"><u><span
-
fusion studies confirmed that ChvI gene was induced by
+
style="color: blue;"> BBa_R0010</span></u></a><br style="">
-
acidic conditions (1). Also, it has been known to implicate in
+
<!--[endif]--></i></p>
-
virulence (1).
+
<u1:p></u1:p>
-
This gene is one of the candidates to be use in our biological pH
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
sensor as a
+
<hr align="center" size="2" width="100%"></div>
-
promoter.</small></span></small><span
+
<p class="MsoNormal" style=""><a name="His"></a><b>6-His Tag</b>: The
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
6-Histidine Tag serves as a tag for Western
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
Blotting if our fluorescent reporters are not expressed as highly as we
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
would like. <br>
-
<hr align="center" size="2" width="100%"></span></div>
+
<i>Note: We are using this tag as an additional method for assay beside
-
<p class="MsoNormal" style=""><b><span
+
fluorescence of GFP and Luciferase.</i> </p>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
name="katA"></a>KatA promoter</span></b><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<br>
-
:</span><small><span style="font-size: 13pt; line-height: 115%;"><small>This
+
more information go to: <a
-
Chromosomal gene is located on the linear chromosome (2) and it seems
+
href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&amp;group=UC_Davis"><i><u><span
-
to be
+
style="color: blue;">UCDAVIS_Parts</span></u></i></a> <u1:p></u1:p>
-
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&nbsp; gene expression to low pH "(2). This gene
+
-
has become a candidate to complete our pH sensor device from this
+
-
evidence.</small></span></small><span
+
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
-
<hr align="center" size="2" width="100%"></span></div>
+
-
<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</span></b><span
+
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
+
-
</span><span style="font-size: 13pt; line-height: 115%;"><small>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 <i>Agrobacterium</i> <i>tumorigenesis </i>(2). Therefore, we
+
-
have chosen
+
-
this gene to be one of our candidates to complete our pH sensor device.</small></span><span
+
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
-
<hr align="center" size="2" width="100%"></span></div>
+
-
<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</span></b><span
+
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:
+
-
</span><small><small><span style="font-size: 13pt; line-height: 115%;"><small>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.</small></span></small></small><span
+
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
+
-
<div class="MsoNormal" style="text-align: center;" align="center"><span
+
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
-
<hr align="center" size="2" width="100%"></span></div>
+
-
<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:</span></b>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.</p>
+
-
<p class="MsoNormal"><small><small><b><span
+
-
style="font-size: 13pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></b><font
+
-
size="-1"><span style="font-size: 13pt; line-height: 115%;"></span></font></small></small><b><u><span
+
-
style="font-size: 12pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></u></b></p>
+
-
<p class="MsoNormal"><span
+
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p>&nbsp;</o:p></span></p>
+
<hr style="width: 100%; height: 2px;"><small style="font-weight: bold;"><span
<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;;"></span></small></div>
style="font-size: 18pt; line-height: 115%; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></small></div>

Latest revision as of 01:22, 15 December 2009

PARTS1

 
Parts:   
Parts related to secretion:                                                                                                                                 Parts related to pH sensor:
Proteins:
Promoters:
Others:
 New parts:
      Promoters:
Proteins:
 

New parts: 

INPNC: The ice-nucleation protein (INP) from Pseudomonas syringae (9) is used by its natural host to nucleate ice formation and is implicated in P.syringae-associated pathogenesisINP and a truncated derivative lacking the central domain (INPNC) have been used extensively for displaying proteins on the surface of E. coli (7).  For instance, AldO and PhaZ1 have been successfully displayed on the surface of E.coli using INPNC (7, 15).
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).  This part was synthesized by Mr. Gene (Regensburg, Germany) with codon optimization and subsequently transferred into vector ( pSB1AK3). 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 BBF RCF-12 standard. 
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. 
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 BBa_K265008.


SS: The signal sequence (SS) for the phaZ1 gene product of Paucimonas lemoignei, a polyhydroxybutyrate depolymerase (15).  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 phaZ1 gene (including SS) and a truncated ice nucleation protein from Pseudomonas syringae (BBa_K265008), could lead to stable expression and secretion of the phaZ1 gene product. 
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 (BBa_K265008) or OmpA (BBa_K103006). 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. 
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 BBa_K265002.

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 (BBa_K265008), could lead to stable expression and secretion of the phaZ1 gene product (15).
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 BBa_K265008 (INPNC) and BBa_K265002 (SS) which is compatible with the BBF RFC-12 Standard.
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 BBa_K265008
We have submitted this part to the part registry in the BBF RFC-12 Standard as part BBa_K265009.

OmpA+SS: Since OmpA is believed to function similarly to INPNC and Park et al. have showed that the fusion of the complete phaZ1 gene (including SS) and a truncated ice nucleation protein from Pseudomonas syringae (BBa_K265008), could lead to stable expression and secretion of the phaZ1 gene product (15), we have decided to test and see if OmpA's ability to secret increases when it is used with a signal sequence.
We have modified this protein to be consistent with BBF RFC-12 Standard and have submitted this part to the part registry, BBa_K265011.


OmpA: OmpA is one of the proteins on the outer membrane of E. coli (13),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)
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” (10)

For more information go to: BBa_K103006


RBS:  Ribosome Binding site number 32 (BBa_J61132) from the registry is being used in our secretion system.
For more information go to: BBa_J61132


Terminator: We are using BBa_B0015, a double terminator, as our terminator in both our secretion and pH system.
For more information go to: BBa_B0015


GFP (Green Fluorescent Protein): 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: BBa_K265003 


Luciferase: Luciferase is a firefly protein that also fluoresces, so it serves as a reporter as well as a testable large protein.
For more information go to: BBa_1712019


LacI: An inducible promoter that was found in the part registry.
For more information go to: BBa_R0010


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.
Note: We are using this tag as an additional method for assay beside fluorescence of GFP and Luciferase.



more information go to: UCDAVIS_Parts