Team:UC Davis/Adding secretion/model 4

From 2009.igem.org

(Difference between revisions)
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<hr style="width: 100%; height: 2px;"><big><big><big><span
<hr style="width: 100%; height: 2px;"><big><big><big><span
style="text-decoration: underline; font-weight: bold;"><small>Secretion
style="text-decoration: underline; font-weight: bold;"><small>Secretion
-
Model
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Model 2:<br>
-
1:<br>
+
<br>
<br>
</small></span></big></big></big><big><span style="font-style: italic;">Click
</small></span></big></big></big><big><span style="font-style: italic;">Click
Line 132: Line 131:
<big><big><big><span
<big><big><big><span
style="text-decoration: underline; font-weight: bold;"></span></big></big></big>
style="text-decoration: underline; font-weight: bold;"></span></big></big></big>
-
<hr style="width: 100%; height: 2px;"><b><span
+
<hr style="width: 100%; height: 2px;">
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style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
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<p class="MsoNormal" style=""><b><span style=""><a name="INPNC"></a>INPNC:
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name="INPNC"></a>INPNC:</span></b><span
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</span></b><span style="">The
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Ice-nucleation
+
ice-nucleation protein (INP) from <i>Pseudomonas syringae</i> is used
-
protein (INP) from Pseudomonas
+
by its
-
Syringae was suggested to be used for display of foreign proteins on
+
natural host to nucleate ice formation and is implicated in<i> P.
-
the
+
syringae</i>
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surface of <i>E. coli</i>(7).Furthermore, researches have shown that
+
associated pathogenesis<i>.&nbsp; </i>INP and a truncated derivative
-
an INP
+
lacking
-
derivative constituting the N-and C-terminal domains can and has been
+
the central domain (INPNC) have been used extensively for displaying
-
used to
+
proteins
-
display foreign proteins on the surface of <i>E. coli</i>(9). In our
+
on the surface of <i>E. coli (7)</i>.&nbsp; For instance, AldO and
-
project we
+
PhaZ1 have
-
are intending to harness and make use of this feature by fusing a
+
been successfully displayed on the surface of <i>E. coli </i>using
-
specific
+
INPNC (7,
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protein to it. <o:p></o:p></span><br>
+
15). <br>
-
<p class="MsoNormal" style="line-height: normal;"><i><span
+
<u1:p></u1:p>Park <i>et al.</i> have shown that INPNC when fused to
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
the <i>phaZ1</i>
-
have
+
gene, including its signal sequence, can serve as a suitable surface
-
modified this protein to Biobrick standard, Tom Knights Standard.<br>
+
delivery
-
For more information go to: <a
+
and secretion device of the otherwise toxic <i>phaZ1</i> gene product<i>
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008">BBa_K265008</a>&nbsp;</span></i><span
+
</i>(15).&nbsp;
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span></p>
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<u1:p></u1:p>This part was synthesized by Mr. Gene (Regensburg,
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<div class="MsoNormal"
+
Germany) with
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
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codon optimization and subsequently transferred into vector (<span
-
align="center"><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
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
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style="-moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial; background-attachment: scroll;">part
 +
name for AK vector).&nbsp;</span></span> 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>
 +
<i><u1:p></u1:p>We have modified this protein to be consistent with 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>.</i> <o:p></o:p></span></p>
 +
<u2:p></u2:p>
 +
<div class="MsoNormal" style="text-align: center;" align="center"><span
 +
style="">
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
-
<p class="MsoNormal" style="line-height: normal;"><a name="OmpA"></a><b><span
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<p class="MsoNormal" style=""><b><span style=""><a name="OmpA"></a>OmpA</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA</span></b><span
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style="">: OmpA
-
style="font-size: 18pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
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is one of the proteins on the outer membrane of <i>E. coli</i> (13),it
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">&nbsp;</span><span
+
is used
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
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as a displaying fusion protein on the cell surface . This part has
-
is one of the proteins on the outer
+
already been
-
membrane of <i>E. coli</i> (13). OmpA has been found to be useful as
+
documented on the parts registry; however, it has not been tested as a
-
utilizable
+
compnent
-
fusion part that can fuse our protein to and display on the surface of <i>E.
+
of secretion system (via fusion with a target protein linked with a
-
coli</i>. 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
cleavable
-
signal sequence. <o:p></o:p></span></p>
+
signal sequence) <i><br>
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<p class="MsoNormal" style="line-height: normal;"><i><span
+
<u1:p></u1:p>We have modified this protein to be consistent with BBF
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
RFC-12 Standard.<br>
-
have
+
Note: “It has remained essentially unknown how proteins of E. coli
-
modified this protein to Biobrick standard, Tom Knights Standard.</span></i><span
+
outer
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span><br>
+
membrane are sorted and incorporated into this membrane” (10)</i> <i><br>
-
<i><span
+
For more information go to:<a
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
+
-
“It has
+
-
remained essentially unknown how proteins of E. coli outer membrane are
+
-
sorted
+
-
and incorporated into this membrane” (10)</span></i><span
+
-
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">
href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006">
-
BBa_K103006</a></span></i><span
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BBa_K103006</a></i>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <a
+
<o:p></o:p></span></p>
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836"><i><span
+
<u2:p></u2:p>
-
style="color: blue;"></span></i></a><o:p></o:p></span></p>
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
<div class="MsoNormal"
+
style="">
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
-
align="center"><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
-
<p class="MsoNormal" style="line-height: normal;"><a name="RBS"></a><b><span
+
<p class="MsoNormal" style=""><b><span style=""><a name="RBS"></a>RBS</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">RBS</span></b><span
+
style="">:&nbsp; Ribosome
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:&nbsp;
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Binding site number 32 (BBa_J61132) from the registry is being used in
-
</span><span
+
our
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Ribosome
+
secretion system. <u2:p></u2:p><br>
-
Binding site number 32 (BBa_J61132)
+
<i>For more information go to:</i> <a
-
from the registry is being used in our secretion system. <o:p></o:p></span><br>
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i>BBa_J61132</i></a><o:p></o:p></span></p>
-
<i><span
+
<u2:p></u2:p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
more
+
style="">
-
information go to:</span></i><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <big><a
+
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><small><span
+
-
style="font-style: italic;">BBa_J61132</span></small></a></big><a
+
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i><span
+
-
style="color: blue;"></span></i></a><o:p></o:p></span></p>
+
-
<div class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
-
align="center"><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
-
<p class="MsoNormal" style="line-height: normal;"><a name="Terminator"></a><b><span
+
<p class="MsoNormal" style=""><b><span style=""><a name="Terminator"></a>Terminator</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Terminator</span></b><span
+
style="">: We are
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
+
using BBa_B0015, a double terminator, as our terminator in both our
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
secretion
-
are using BBa_B0015, a double terminator, as
+
and pH system.<u2:p></u2:p><br>
-
our terminator in both our secretion and pH system.<o:p></o:p></span><br>
+
<i>For more information go to:</i> <a
-
<i><span
+
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i>BBa_B0015</i></a>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">For
+
<o:p></o:p></span></p>
-
more
+
<u2:p></u2:p>
-
information go to:</span></i><span
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <a
+
style="">
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><span
+
-
style="font-style: italic;">BBa_B0015</span></a> <a
+
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i><span
+
-
style="color: blue;"></span></i></a><o:p></o:p></span></p>
+
-
<div class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
-
align="center"><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
-
<p class="MsoNormal" style="line-height: normal;"><a name="GFP"></a><b><span
+
<p class="MsoNormal" style=""><b><span style=""><a name="GFP"></a>GFP</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">GFP</span></b><span
+
style="">: We are
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
+
using Green Fluorescent Protein as a reporter that also serves as a
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
small
-
are using Green Fluorescent Protein as a
+
protein in testing our secretion system.<br>
-
reporter that also serves as a small protein in testing our secretion
+
<i>For more informaiton go to: </i><a
-
system.<br>
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003"><i>BBa_K265003</i></a><i>&nbsp;</i><u2:p></u2:p><o:p></o:p></span></p>
-
<span style="font-style: italic;">For more informaiton go to: </span><a
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
style="font-style: italic;"
+
style="">
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003">BBa_K265003</a><span
+
-
style="font-style: italic;">&nbsp;</span><o:p></o:p></span></p>
+
-
<div class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
-
align="center"><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
-
<p class="MsoNormal" style="line-height: normal;"><a name="Luciferase"></a><b><span
+
<p class="MsoNormal" style=""><b><span style=""><a name="Luciferase"></a>Luciferase</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Luciferase</span></b><span
+
style="">: Luciferase
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
+
is a firefly protein that also fluoresces, so it serves as a reporter
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Luciferase
+
as well
-
is a firefly protein that also
+
as a testable large protein.<br>
-
fluoresces, so it serves as a reporter as well as a testable large
+
<i>For more information go to: <a
-
protein.<br>
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019">BBa_1712019</a></i>
-
<span style="font-style: italic;">For more information go to: <a
+
<u2:p></u2:p><o:p></o:p></span></p>
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019">BBa_1712019</a></span>
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
<o:p></o:p></span></p>
+
style="">
-
<div class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
-
align="center"><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
-
<p class="MsoNormal" style="line-height: normal;"><a name="LacI"></a><b><span
+
<p class="MsoNormal" style=""><b><span style=""><a name="LacI"></a>LacI</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">LacI</span></b><span
+
style="">: One
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">: </span><span
+
inducible Promoter which was found in the part registry.<br>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">One
+
<i>For more information go to:<a
-
inducible Promoter which was found in the
+
-
part registry.<br>
+
-
</span><i><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i><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_R0010">
href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010">
-
BBa_R0010</a><br>
+
BBa_R0010</a><br style="">
-
</span></i><span
+
<!--[endif]--></i><o:p></o:p></span></p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
+
<u2:p></u2:p>
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010"><i><span
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
style="color: blue;"></span></i></a><o:p></o:p></span></p>
+
style="">
-
<div class="MsoNormal"
+
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
-
align="center"><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
-
<p class="MsoNormal" style="line-height: normal;"><a name="SS"></a><b><span
+
<p class="MsoNormal" style=""><b><span style=""><a name="SS"></a>SS</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS</span></b><span
+
style="">:The
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
+
signal sequence (SS) for the <i>phaZ1 </i>gene product of <i>Paucimonas
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">This
+
lemoignei</i>, a polyhydroxybutyrate depolymerase (15).&nbsp; In the
-
signal sequence, when placed between
+
native
-
INPNC, contains a cleavable site that allows the target fusion protein
+
protein the signal sequence is cleaved between residues Ala37 and
-
to
+
Leu38.&nbsp;
-
‘secrete’ from INPNC. We will do the same with OmpA. <o:p></o:p></span><br>
+
Park <i>et al. </i>have showed that the fusion of the complete <i>phaZ1
-
<i><span
+
</i>gene
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
(including SS) and a truncated ice nucleation protein from <i>Pseudomonas
-
have
+
syringae</i> (<a
-
modified this protein to Biobrick standard, Tom Knights Standard.<br>
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span
-
For more infromation go to :<a
+
style="color: rgb(0, 0, 153);">BBa_K265008</span></i></a>), could lead
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002">BBa_K265002</a></span></i><span
+
to stable
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <o:p></o:p></span></p>
+
expression and secretion of the <i>phaZ1</i> gene product.&nbsp; <br>
-
<div class="MsoNormal"
+
<u1:p></u1:p>We propose that the signal sequence might be generally
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
useful as a
-
align="center"><span
+
cleavage tag in secretion systems that include a membrane anchor
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
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">BBa_K103006</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>
 +
<i><u1:p></u1:p>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>. <o:p></o:p></span></p>
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<u2:p></u2:p>
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<div class="MsoNormal" style="text-align: center;" align="center"><span
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style="">
<hr align="center" size="2" width="100%"></span></div>
<hr align="center" size="2" width="100%"></span></div>
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<a name="His"></a><b><span
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<p class="MsoNormal"><b><span style=""><a name="Tag"></a>6-His Tag</span></b><span
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">6-His
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style="">:The 6-Histidine Tag serves as a tag for
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Tag</span></b><span
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Western Blotting if our fluorescent reporters are not expressed as
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style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
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highly as we
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">The
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would like. <u2:p></u2:p><br>
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6-Histidine Tag serves as a tag for Western
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<i>Note: We are using this tag, just in case if the GFP or Luciferase
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Blotting if our fluorescent reporters are not expressed as highly as we
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does not
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would
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work under a plate reader.</i></span></p>
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like. <o:p></o:p></span><br>
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<p class="MsoNormal"><span style=""></span></p>
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<small><i><span
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<hr style="width: 100%; height: 2px;">
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
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<p class="MsoNormal"><span style="">&nbsp;<o:p></o:p></span></p>
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We are
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using this tag, just in case if the GFP or Luciferase does not work
+
-
under a
+
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plate reader.<br>
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</span></i></small>
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<hr style="width: 100%; height: 2px;"><small><i><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i></small>
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<p class="MsoNormal"><br>
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<span
<span

Revision as of 16:43, 21 October 2009

Model 1

=

Secretion Model 2:

Click on an individual part for more information.


a.

b.


INPNC: The ice-nucleation protein (INP) from Pseudomonas syringae 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 INPNC when fused to the phaZ1 gene, including its signal sequence, 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 (part name for AK vector).  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 BBF RFC-12 Standard. We have submitted this part to the parts registry as part BBa_K265008.


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 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: We are using Green Fluorescent Protein as a reporter that 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: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.


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.