Team:UC Davis/Adding secretion/model 1

From 2009.igem.org

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alt="" src="https://static.igem.org/mediawiki/2009/0/0c/UCDAVIS_GENE1.png"><a
alt="" src="https://static.igem.org/mediawiki/2009/0/0c/UCDAVIS_GENE1.png"><a
-
href="#His"><img alt=""
+
href="#Tag"><img alt=""
src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
style="border: 0px solid ; width: 134px; height: 66px; background-color: rgb(255, 255, 255);"></a><a
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src="https://static.igem.org/mediawiki/2009/7/7d/UCDAVIS_GFP.png"
src="https://static.igem.org/mediawiki/2009/7/7d/UCDAVIS_GFP.png"
style="border: 0px solid ; width: 83px; height: 55px; background-color: rgb(255, 255, 255);"></a><a
style="border: 0px solid ; width: 83px; height: 55px; background-color: rgb(255, 255, 255);"></a><a
-
href="#His"><img alt=""
+
href="#Tag"><img alt=""
src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
style="border: 0px solid ; width: 110px; height: 58px; background-color: rgb(255, 255, 255);"></a><a
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src="https://static.igem.org/mediawiki/2009/9/98/UCDAVIS_Luciferase.png"
src="https://static.igem.org/mediawiki/2009/9/98/UCDAVIS_Luciferase.png"
style="border: 0px solid ; width: 87px; height: 49px;"></a><a
style="border: 0px solid ; width: 87px; height: 49px;"></a><a
-
href="#His"><img alt=""
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href="#Tag"><img alt=""
src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
src="https://static.igem.org/mediawiki/2009/c/c0/UCDAVIS_6HIS.png"
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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;">
<hr style="width: 100%; height: 2px;">
-
<p class="MsoNormal" style="line-height: normal;"><b><span
+
<p class="MsoNormal" style=""><b><span style=""><a name="INPNC"></a>INPNC:
-
style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
+
</span></b><span style="">The
-
name="INPNC"></a></span></b><b>INPNC:</b>The ice-nucleation protein
+
ice-nucleation protein (INP) from <i>Pseudomonas syringae</i> is used
-
(INP) from <i>Pseudomonas
+
by its
-
syringae</i> is used by its natural host to nucleate ice formation and
+
natural host to nucleate ice formation and is implicated in<i>
-
is
+
P.syringae</i>-associated pathogenesis<i>.&nbsp; </i>INP, as well as a
-
implicated in<i> P. syringae</i> associated pathogenesis<i>.&nbsp; </i>INP
+
truncated derivative
-
and
+
lacking
-
a truncated derivative lacking the central domain (INPNC) have been
+
the central domain (INPNC), have been used extensively for displaying
-
used
+
proteins
-
extensively for displaying proteins on the surface of <i>E. coli (7)</i>.&nbsp;
+
on the surface of <i>E. coli (7)</i>.&nbsp; For instance, AldO and
-
For instance, AldO and PhaZ1 have been successfully displayed on the
+
PhaZ1 have
-
surface of
+
been successfully displayed on the surface of <i>E. coli </i>using
-
<i>E. coli </i>using INPNC (7, 15). <br>
+
INPNC (7,
-
<u1:p></u1:p>Park <i>et al.</i> have shown that INPNC when fused to
+
15). <br>
 +
<u1:p></u1:p>Park <i>et al.</i> have shown that INPNC, when fused to
the <i>phaZ1</i>
the <i>phaZ1</i>
gene, including its signal sequence, can serve as a suitable surface
gene, including its signal sequence, can serve as a suitable surface
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Germany) with
Germany) with
codon optimization and subsequently transferred into vector (<span
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;">part
+
style="background-image: none; background-repeat: repeat; background-attachment: scroll; background-position: 0% 50%; color: black; -moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial;"><span
-
name for AK vector).&nbsp;</span>
+
style="-moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial; background-attachment: scroll;"><a
-
As it is expected that this part will be used in the context of the
+
href="http://partsregistry.org/Part:pSB1AK3">pSB1AK3</a>).</span></span>
-
fusion
+
As it is expected that this
-
protein, the prefix and suffix for this part are consistent with the <i>BBF
+
part will be used in the context of the fusion protein, the prefix and
-
RCF-12</i> standard.&nbsp; <br>
+
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
<u1:p></u1:p>We have proposed to build and test a general protein
secretion
secretion
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secrete
secrete
any target protein.&nbsp; <br>
any target protein.&nbsp; <br>
-
<u1:p></u1:p><i>We have modified this protein to be consistent with BBF
+
<i><u1:p></u1:p>We have modified this protein to be consistent with the
 +
BBF
RFC-12
RFC-12
Standard. We have submitted this part to the parts registry as part </i><a
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
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><i><span
+
style="color: rgb(0, 0, 153);">BBa_K265008</span></i></a><i>.</i> <o:p></o:p></span></p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i><span
+
<u2:p></u2:p>
-
style="font-size: 12pt; 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
-
<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="OmpA"></a><b><span
+
<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
+
style="">: OmpA
-
style="font-size: 18pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
+
is a protein found on the outer membrane of <i>E. coli</i> (13) and
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </span>OmpA
+
is used
-
is one of the
+
as a displaying fusion protein on the cell surface. This part has
-
proteins on
+
already been
-
the outer membrane of <i>E. coli</i> (13),it is used as a displaying
+
documented on the parts registry; however, it has not been tested as a
-
fusion
+
component
-
protein on the cell surface . This part has already been documented on
+
of a secretion system (via fusion with a target protein linked with a
-
the parts
+
cleavable
-
registry; however, it has not been tested as a compnent of secretion
+
signal sequence). <i><br>
-
system
+
<u1:p></u1:p>We have modified this protein to be consistent with the
-
(via fusion with a target protein linked with a cleavable signal
+
BBF
-
sequence) <i><br>
+
RFC-12 Standard.<br>
-
<u1:p></u1:p>We have modified this protein to be consistent with BBF
+
-
RFC-12
+
-
Standard.<br>
+
Note: “It has remained essentially unknown how proteins of E. coli
Note: “It has remained essentially unknown how proteins of E. coli
outer
outer
membrane are sorted and incorporated into this membrane” (10)</i> <i><br>
membrane are sorted and incorporated into this membrane” (10)</i> <i><br>
For more information go to:<a
For more information go to:<a
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"><u><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006">
-
style="color: blue;"> BBa_K103006</span></u></a></i><i><span
+
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/Part:BBa_K103006"></a></span></i><span
+
<u2:p></u2:p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <a
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J36836"><i><span
+
style="">
-
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="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;
+
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="">: This is an
-
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><big><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></big>The
+
signal sequence (SS) for the <i>phaZ1 </i>gene product of <i>Paucimonas
signal sequence (SS) for the <i>phaZ1 </i>gene product of <i>Paucimonas
lemoignei</i>, a polyhydroxybutyrate depolymerase (15).&nbsp; In the
lemoignei</i>, a polyhydroxybutyrate depolymerase (15).&nbsp; In the
Line 330: Line 284:
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span
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
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
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006">BBa_K103006</a>).<span
-
style="color: blue;">BBa_K103006</span></a>).<span
+
style="color: rgb(0, 41, 57);"> </span></i>The proposed constructs
-
style="color: rgb(0, 41, 57);"> </span></i>The
+
would consist of a
-
proposed constructs would consists of a membrane anchor (INPNC or OmpA)
+
membrane anchor (INPNC or OmpA) followed by the cleavable signal
-
followed by the cleavable signal sequence and finally a target protein
+
sequence and
-
marked
+
finally a target protein marked for secretion.&nbsp; <br>
-
for secretion.&nbsp; <br>
+
<i><u1:p></u1:p>Since we expect that this part will be used in the
-
<u1:p></u1:p><i>Since we expect that this part will be used in the
+
context of a
context of a
-
fusion protein, we have modified this protein to be consistent with BBF
+
fusion protein, we have modified this protein to be consistent with the
 +
BBF
RFC-12
RFC-12
Standard. We have submitted this part to the part registry as part </i><a
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
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"><i><span
-
style="color: rgb(0, 0, 153);">BBa_K265002</span></i></a>.<span
+
style="color: rgb(0, 0, 153);">BBa_K265002</span></i></a>. <o:p></o:p></span></p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span><i><span
+
<u2:p></u2:p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
+
<div class="MsoNormal" style="text-align: center;" align="center"><span
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"></a></span></i><span
+
style="">
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <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>
-
<a name="His"></a><b><span
+
<p class="MsoNormal"><b><span style=""><a name="Tag"></a>6-His Tag</span></b><span
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">6-His
+
style="">: The 6-Histidine Tag serves as a tag for
-
Tag</span></b><span
+
Western Blotting if our fluorescent reporters are not expressed as
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
+
highly as we
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">The
+
would like. <u2:p></u2:p><br>
-
6-Histidine Tag serves as a tag for Western
+
<i>Note: We are using this tag just in case the GFP or Luciferase
-
Blotting if our fluorescent reporters are not expressed as highly as we
+
do not
-
would
+
work under a plate reader.</i> <o:p></o:p></span></p>
-
like. <o:p></o:p></span><br>
+
-
<small><i><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Note:
+
-
We are
+
-
using this tag, just in case if the GFP or Luciferase does not work
+
-
under a
+
-
plate reader.</span></i></small>
+
<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;"

Latest revision as of 23:26, 21 October 2009

Model 1

=

Secretion Model 1:

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, as well as 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 (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.


OmpA: OmpA is a protein found on the outer membrane of E. coli (13) and 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 component of a secretion system (via fusion with a target protein linked with a cleavable signal sequence).
We have modified this protein to be consistent with the 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: This is an 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 consist 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 the 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 the GFP or Luciferase do not work under a plate reader.