Team:UC Davis/Parts

From 2009.igem.org

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<hr style="width: 100%; height: 2px;">
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<p class="MsoNormal" style="line-height: normal;"><b><span
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<p class="MsoNormal" style="line-height: normal;"><a name="INPNC"></a><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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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">INPNC:
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name="INPNC"></a>INPNC:</span></b><span
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</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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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">An
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protein (INP) from Pseudomonas
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exogenous gene (Ice Nucleation Protein)
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Syringae was suggested to be used for display of foreign proteins on
+
found to be expressed on the surface of E.Coli.&nbsp; (The NC stands
 +
for the
 +
shortened version of the entire gene, which works perfectly in E.Coli).
 +
Ice-nucleation protein (INP) from Pseudomonas Syringae was suggested to
 +
be used
 +
for display of foreign proteins on the surface of <i>E. coli</i>(7).
 +
Furthermore, researches have shown that an INP derivative constituting
the
the
-
surface of <i>E. coli</i>(7).Furthermore, researches have shown that
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N-and C-terminal domains can and has been used to display foreign
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an INP
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proteins on
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derivative constituting the N-and C-terminal domains can and has been
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the surface of <i>E. coli</i>(9). In our project we are intending to
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used to
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harness
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display foreign proteins on the surface of <i>E. coli</i>(9). In our
+
and make use of this feature by fusing a specific protein to it. <br>
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project we
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<i>We have modified this protein to Biobrick standard, Tom Knights
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are intending to harness and make use of this feature by fusing a
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Standard.<br>
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specific
+
-
protein to it. <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
+
-
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
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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href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><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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style="color: blue;">BBa_K265008</span></a>&nbsp;</i> <o:p></o:p></span></p>
<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA</span></b><span
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA</span></b><span
style="font-size: 18pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
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: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </span><span
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
is one of the proteins on the outer
is one of the proteins on the outer
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utilizable
utilizable
fusion part that can fuse our protein to and display on the surface of <i>E.
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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coli</i>.and readily available as a biobrick part to test it's efficacy
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however,
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versus
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it has not been tested via fusion with a target protein linked with a
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INP in fusion to SS. This part has already been documented on the parts
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cleavable
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registry; however, it has not been tested via fusion with a target
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signal sequence. <o:p></o:p></span></p>
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protein
 +
linked with a cleavable signal sequence. <o:p></o:p></span></p>
<p class="MsoNormal" style="line-height: normal;"><i><span
<p class="MsoNormal" style="line-height: normal;"><i><span
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
have
have
modified this protein to Biobrick standard, Tom Knights Standard.</span></i><span
modified this protein to Biobrick standard, Tom Knights Standard.</span></i><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span><br>
+
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <br>
-
<i><span
+
<i>Note: “It has remained essentially unknown how proteins of E. coli
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
+
outer
-
“It has
+
membrane are sorted and incorporated into this membrane” (10)</i> <br>
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remained essentially unknown how proteins of E. coli outer membrane are
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<i>For more information go to:<a
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sorted
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href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"><span
-
and incorporated into this membrane” (10)</span></i><span
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style="color: blue;"> BBa_K103006</span></a></i> <o:p></o:p></span></p>
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span><br>
+
-
<i><span
+
-
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_K103006">
+
-
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="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Ribosome
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Ribosome
Binding site number 32 (BBa_J61132)
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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from the registry is being used in our secretion system. <br>
-
<i><span
+
<i>For more information go to:</i> </span><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
+
style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
-
more
+
-
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
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>
+
style="font-size: 12pt; color: blue;">BBa_J61132</span></i></a></span><span
 +
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></p>
<div class="MsoNormal"
<div class="MsoNormal"
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
are using BBa_B0015, a double terminator, as
are using BBa_B0015, a double terminator, as
-
our terminator in both our secretion and pH system.<o:p></o:p></span><br>
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our terminator in both our secretion and pH system.<br>
-
<i><span
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<i>For more information go to:</i> <a
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
+
-
more
+
-
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
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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style="color: blue;">BBa_B0015</span></i></a> <o:p></o:p></span></p>
<div class="MsoNormal"
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<p class="MsoNormal" style="line-height: normal;"><a name="GFP"></a><b><span
<p class="MsoNormal" style="line-height: normal;"><a name="GFP"></a><b><span
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">GFP</span></b><span
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: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <b>(</b></span><b><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Green
-
are using Green Fluorescent Protein as a
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Fluorescent Protein)</span></b><span
-
reporter that also serves as a small protein in testing our secretion
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> :
-
system.<br>
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Superfolder, which will let this protein
-
<span style="font-style: italic;">For more informaiton go to: </span><a
+
fold quickly so we can use either a fluorescent reader or UV light to
-
style="font-style: italic;"
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detect.
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href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003">BBa_K265003</a><span
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It also serves as a small protein in testing our secretion system.<br>
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style="font-style: italic;">&nbsp;</span><o:p></o:p></span></p>
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<i>For more informaiton go to: </i><a
 +
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003"><i><span
 +
style="color: blue;">BBa_K265003</span></i></a><i>&nbsp;</i><o:p></o:p></span></p>
<div class="MsoNormal"
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style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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fluoresces, so it serves as a reporter as well as a testable large
fluoresces, so it serves as a reporter as well as a testable large
protein.<br>
protein.<br>
-
<span style="font-style: italic;">For more information go to: <a
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<i>For more information go to: <a
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019">BBa_1712019</a></span>
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href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019"><span
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<o:p></o:p></span></p>
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style="color: blue;">BBa_1712019</span></a></i> <o:p></o:p></span></p>
<div class="MsoNormal"
<div class="MsoNormal"
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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inducible Promoter which was found in the
inducible Promoter which was found in the
part registry.<br>
part registry.<br>
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</span><i><span
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<i>For more information go to:<a
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i><i><span
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href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010"><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
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style="color: blue;"> BBa_R0010</span></a><br style="">
-
more information go to:<a
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<!--[if !supportLineBreakNewLine]--><br style="">
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href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010">
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<!--[endif]--></i><o:p></o:p></span></p>
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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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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS</span></b><span
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS</span></b><span
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">This
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">It
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signal sequence, when placed between
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allows protein fused to outer membrane
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INPNC, contains a cleavable site that allows the target fusion protein
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proteins to become cleaved free. In this experiment this signal
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to
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sequence when
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‘secrete’ from INPNC. We will do the same with OmpA. <o:p></o:p></span><br>
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placed between INPNC, contains a cleavable site that allows the target
-
<i><span
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fusion
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
protein to ‘secrete’ from INPNC. We will do the same with OmpA. <br>
-
have
+
<i>We have modified this protein to Biobrick standard, Tom Knights
-
modified this protein to Biobrick standard, Tom Knights Standard.<br>
+
Standard.<br>
For more infromation go to :<a
For more infromation go to :<a
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002">BBa_K265002</a></span></i><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span></p>
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style="color: blue;">BBa_K265002</span></a></i> <o:p></o:p></span></p>
<div class="MsoNormal"
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Blotting if our fluorescent reporters are not expressed as highly as we
Blotting if our fluorescent reporters are not expressed as highly as we
would
would
-
like. <o:p></o:p></span><br>
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like. <br>
-
<small><i><span
+
<i>Note: We are using this tag, just in case if the GFP or Luciferase
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
+
does not
-
We are
+
work under a plate reader.</i> <o:p></o:p></span></p>
-
using this tag, just in case if the GFP or Luciferase does not work
+
-
under a
+
-
plate reader.</span></i></small><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span></p>
+
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was induced by acidic conditions (1), therefore, this is one of our
was induced by acidic conditions (1), therefore, this is one of our
candidates
candidates
-
to complete our pH sensor device.</span><span
+
to complete our pH sensor device. <o:p></o:p></span></p>
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span> <br>
+
-
</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>

Revision as of 03:11, 13 October 2009

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

INPNC: An exogenous gene (Ice Nucleation Protein) found to be expressed on the surface of E.Coli.  (The NC stands for the shortened version of the entire gene, which works perfectly in E.Coli). 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). In our project we are intending to harness and make use of this feature by fusing a specific protein to it.
We have modified this protein to Biobrick standard, Tom Knights Standard.
For more information go to: BBa_K265008 


OmpA: 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.and readily available as a biobrick part to test it's efficacy versus INP in fusion to SS. 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.

We have modified this protein to Biobrick standard, Tom Knights Standard.
Note: “It has remained essentially unknown how proteins of E. coli outer membrane are sorted and incorporated into this membrane” (10)
For more information go to: BBa_K103006


RBSRibosome 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) : Superfolder, which will let this protein fold quickly so we can use either a fluorescent reader or UV light to detect. 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: One inducible Promoter which was found in the part registry.
For more information go to: BBa_R0010


SS:It allows protein fused to outer membrane proteins to become cleaved free. In this experiment 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.
We have modified this protein to Biobrick standard, Tom Knights Standard.
For more infromation go to :BBa_K265002


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, just in case if the GFP or Luciferase does not work under a plate reader.


ChvI promoter: 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.


KatA promoter :This 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 Agrobacterium tumorigenesis (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.


AopB promoter: 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.


PhoA promoter: 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.


ImpA promoter: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.