Team:UC Davis/Parts

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<p class="MsoNormal"
<p class="MsoNormal"
style="line-height: normal; font-weight: bold; text-align: center; text-decoration: underline;"><big><big>New
style="line-height: normal; font-weight: bold; text-align: center; text-decoration: underline;"><big><big>New
-
parts:</big></big></p>
+
parts:</big></big>&nbsp;</p>
-
<p class="MsoNormal" style="line-height: normal;"><a name="INPNC"></a><b><span
+
<span style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span>
-
style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">INPNC:
+
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
-
</span></b><span
+
name="INPNC"></a><b>INPNC:</b> The ice-nucleation protein (INP) from <i>Pseudomonas
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">An
+
syringae</i> (<a
-
exogenous gene, Ice-nucleation
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">9</a>)
-
protein (INP) from <i>Pseudomonas syringae</i> was suggested to be
+
is used by its natural host to nucleate ice formation and
-
used for
+
is implicated in<i> P.syringae</i>-associated pathogenesis<i>.&nbsp; </i>INP
-
display of foreign proteins on the surface of <i>E. coli. </i>(7).
+
and
-
Furthermore,
+
a truncated derivative lacking the central domain (INPNC) have been
-
studies have shown that an INP derivative constituting the N-and
+
used
-
C-terminal
+
extensively for displaying proteins on the surface of <i>E. coli </i>(<a
-
domains can and has been used to display foreign proteins on the
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">7</a>).&nbsp;
-
surface of <i>E.
+
For instance, AldO and PhaZ1 have been successfully displayed on the
-
coli. </i>(9).In our project we are intending to harness and make use
+
surface of
-
of this
+
<i>E.coli </i>using INPNC (<a
-
feature by fusing a specific protein to it. <br>
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">7, 15</a>).
-
<i>We have modified this protein to be consistent with BBF RFC-12
+
<br>
-
Standard. We
+
<u1:p></u1:p>Park <i>et al.</i> have shown that when INPNC is fused to
-
have submitted this part to the part registry.<br>
+
the <i>phaZ1</i>
-
For more information go to: <a
+
gene and its signal sequence, it can serve as a suitable surface
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><span
+
delivery
-
style="color: blue;">BBa_K265008</span></a> </i><o:p></o:p><br>
+
and secretion device of the otherwise toxic <i>phaZ1</i> gene product(<a
-
</span></p>
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).&nbsp;
-
<p class="MsoNormal" style="line-height: normal;"><span
+
<u1:p></u1:p>This part was synthesized by Mr. Gene (Regensburg,
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span><a
+
Germany) with
-
name="SS"></a><b><span
+
codon optimization and subsequently transferred into vector (<span
-
style="font-size: 14pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">SS</span></b><b><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: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span></b><span
+
<a href="http://partsregistry.org/Part:pSB1AK3">pSB1AK3</a>). As it is
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </span><span
+
expected that this part will be used in the context of the
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">It
+
fusion
-
allows protein fused to outer
+
protein, the prefix and suffix for this part are consistent with the <i>BBF
-
membrane proteins to become cleaved free. In this experiment this
+
RCF-12</i> standard.&nbsp; <br>
-
signal
+
<u1:p></u1:p>We have proposed to build and test a general protein
-
sequence when placed between INPNC, contains a cleavable site that
+
secretion
-
allows the
+
system modeled after that developed by Park <i>et al. </i>in which a
-
target fusion protein to ‘secrete’ from INPNC. We will do the same with
+
fusion of
-
OmpA. <br>
+
INPNC and the signal sequence from the <i>phaZ1</i> gene are used to
-
<i>We have modified this protein to be consistent with BBF RFC-12
+
secrete
-
Standard. We
+
any target protein.&nbsp; <br>
-
have submitted this part to the part registry.<br>
+
<u1:p></u1:p><i>We have modified this protein to be consistent with the
-
For more information go to: <a
+
BBF
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"><span
+
RFC-12
-
style="color: blue;">BBa_K265002</span></a></i><o:p></o:p></span></p>
+
Standard. We have submitted this part to the parts registry as part </i><a
-
<p class="MsoNormal" style="line-height: normal;"><a name="INPNCSS"></a><b><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span
-
style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">INPNC
+
style="color: rgb(0, 0, 153);">BBa_K265008</span></i></a><i>.<br>
-
+ SS: </span></b><span
+
</i></p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">It
+
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><br>
-
has been suggested that INPNC can
+
<i><u1:p></u1:p></i><o:p></o:p></p>
-
secrete the interest gene better if it is used with the right signal
+
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
-
sequence. Therefore, we are using INPNC and SS toegther in one of our
+
name="SS"></a><b><u1:p></u1:p>SS:</b> The
-
secretion models.<br>
+
signal sequence (SS) for the <i>phaZ1 </i>gene product of <i>Paucimonas
-
<i>We have submitted this part to the part registry.<br>
+
lemoignei</i>, a polyhydroxybutyrate depolymerase (<a
-
For more information go to: <a
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).&nbsp;
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K265009"><span
+
In the
-
style="color: blue;">BBa_K265009</span></a><o:p></o:p></i></span></p>
+
native
-
<p class="MsoNormal" style="line-height: normal;"><a name="OmpAss"></a><b><span
+
protein the signal sequence is cleaved between residues Ala37 and
-
style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
+
Leu38.&nbsp;
-
+ SS: </span></b><span
+
Park <i>et al. </i>have showed that the fusion of the complete <i>phaZ1
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Since
+
</i>gene
-
OmpA is believed to function
+
(including SS) and a truncated ice nucleation protein from <i>Pseudomonas
-
similarly to INPNC and INPNC believed to function better when it is
+
syringae</i> (<a
-
used with
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265008"><i><span
-
the right signal sequence, we have decided to test and see if OmpA's
+
style="color: rgb(0, 0, 153);">BBa_K265008</span></i></a>), could lead
-
ability to
+
to stable
-
secret increases when it is used by a signal sequence.<br>
+
expression and secretion of the <i>phaZ1</i> gene product.&nbsp; <br>
-
<i>We have submitted this part to the part registry.</i><br>
+
<u1:p></u1:p>We propose that the signal sequence might be generally
-
<i>For more information go to: </i><a
+
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>
 +
<u1:p></u1:p>
 +
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
 +
name="INPNCSS"></a><b>INPNC+SS:</b> 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 (<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>
 +
<u1:p></u1:p>
 +
<p class="freeform0" style="margin-bottom: 6pt; line-height: 19pt;"><a
 +
name="OmpAss"></a><b>OmpA+SS:</b> Since OmpA is believed to function
 +
similarly
 +
to INPNC and 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 (<a
 +
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>),
 +
we
 +
have decided
 +
to test and see if OmpA's ability to secret increases when it is used
 +
with a
 +
signal sequence.<br>
 +
<u1:p></u1:p><i>We have modified this protein to be consistent with BBF
 +
RFC-12
 +
Standard and have submitted this part to the part registry, </i><a
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K265011"><i><span
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K265011"><i><span
-
style="color: blue;">BBa_K265011</span></i></a><o:p></o:p></span></p>
+
style="color: rgb(0, 0, 153);">BBa_K265011</span></i><u><span
-
<hr style="width: 100%; height: 2px;"><b><span
+
style="color: blue;">.</span></u></a></p>
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
+
<u1:p></u1:p>
-
name="OmpA"></a>OmpA</span></b><span
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
style="font-size: 18pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:</span><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> </span><span
+
<p class="MsoNormal"><a name="OmpA"></a><b>OmpA</b>: OmpA is one of the
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
+
proteins on
-
is one of the proteins on the outer
+
the outer membrane of <i>E. coli</i> (<a
-
membrane of <i>E. coli</i> (13),it is used as a displaying fusion
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">13</a>),it
 +
is used as a displaying
 +
fusion
protein on the cell surface . This part has already been documented on
protein on the cell surface . This part has already been documented on
the parts
the parts
registry; however, it has not been tested as a compnent of secretion
registry; however, it has not been tested as a compnent of secretion
-
system (via fusion with a target
+
system
-
protein
+
(via fusion with a target protein linked with a cleavable signal
-
linked with a cleavable signal sequence) <o:p></o:p></span><span
+
sequence) <i><br>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i><br>
+
<u1:p></u1:p>We have modified this protein to be consistent with BBF
-
We
+
RFC-12
-
have modified this protein to be consistent with BBF RFC-12 Standard.</i></span><i><span
+
Standard.<br>
-
style="font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><br>
+
Note: “It has remained essentially unknown how proteins of E. coli
-
</span>Note:
+
-
“It has remained essentially unknown how proteins of E. coli
+
outer
outer
-
membrane are sorted and incorporated into this membrane” (10)</i> <span
+
membrane are sorted to and incorporated into this membrane” (<a
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i><br>
+
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">10</a>)</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"><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"><u><span
-
style="color: blue;"> BBa_K103006</span></a></i> <o:p></o:p></span>
+
style="color: blue;"> BBa_K103006</span></u></a></i> </p>
-
<div class="MsoNormal"
+
<u1:p></u1:p>
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
align="center"><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<p class="MsoNormal" style=""><a name="RBS"></a><b>RBS</b>:&nbsp;
-
<hr align="center" size="2" width="100%"></span></div>
+
Ribosome Binding site number 32 (BBa_J61132)
-
<p class="MsoNormal" style="line-height: normal;"><a name="RBS"></a><b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">RBS</span></b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">:&nbsp;
+
-
</span><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Ribosome
+
-
Binding site number 32 (BBa_J61132)
+
from the registry is being used in our secretion system. <br>
from the registry is being used in our secretion system. <br>
-
<i>For more information go to:</i> </span><span
 
-
style="font-size: 13.5pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><a
 
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i><span
 
-
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"
 
-
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>
 
-
<p class="MsoNormal" style="line-height: normal;"><a name="Terminator"></a><b><span
 
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Terminator</span></b><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;;">We
 
-
are using BBa_B0015, a double terminator, as
 
-
our terminator in both our secretion and pH system.<br>
 
<i>For more information go to:</i> <a
<i>For more information go to:</i> <a
-
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i><u><span
-
style="color: blue;">BBa_B0015</span></i></a> <o:p></o:p></span></p>
+
style="color: blue;">BBa_J61132</span></u></i></a></p>
-
<div class="MsoNormal"
+
<u1:p></u1:p>
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
align="center"><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 12pt; 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 class="MsoNormal" style="line-height: normal;"><a name="GFP"></a><b><span
+
terminator, as our terminator in both our secretion and pH system.<br>
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">GFP</span></b><span
+
<i>For more information go to:</i> <a
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <b>(</b></span><b><span
+
href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i><u><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Green
+
style="color: blue;">BBa_B0015</span></u></i></a> </p>
-
Fluorescent Protein)</span></b><span
+
<u1:p></u1:p>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> :
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
Mutant of GFP known to be very stable (superfolder), which will let
+
<hr align="center" size="2" width="100%"></div>
-
this protein
+
<p class="MsoNormal" style=""><a name="GFP"></a><b>GFP</b> <b>(Green
-
fold quickly so we can use either a fluorescent reader or UV light to
+
Fluorescent Protein)</b>: Mutant of GFP
-
detect it. Therefore it has been used as a reporter in our secretion
+
known to be very stable (superfolder), which will let this protein fold
-
system.
+
quickly
-
It also serves as a small protein in testing our secretion system.<br>
+
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.<br>
<i>For more informaiton go to: </i><a
<i>For more informaiton go to: </i><a
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003"><i><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003"><i><u><span
-
style="color: blue;">BBa_K265003</span></i></a><i>&nbsp;</i><o:p></o:p></span></p>
+
style="color: blue;">BBa_K265003</span></u></i></a><i>&nbsp;</i></p>
-
<div class="MsoNormal"
+
<u1:p></u1:p>
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
align="center"><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<p class="MsoNormal" style=""><a name="Luciferase"></a><b>Luciferase</b>:
-
<hr align="center" size="2" width="100%"></span></div>
+
Luciferase is a firefly protein that
-
<p class="MsoNormal" style="line-height: normal;"><a name="Luciferase"></a><b><span
+
also fluoresces, so it serves as a reporter as well as a testable large
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Luciferase</span></b><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;;">Luciferase
+
-
is a firefly protein that also
+
-
fluoresces, so it serves as a reporter as well as a testable large
+
protein.<br>
protein.<br>
<i>For more information go to: <a
<i>For more information go to: <a
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019"><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019"><u><span
-
style="color: blue;">BBa_1712019</span></a></i> <o:p></o:p></span></p>
+
style="color: blue;">BBa_1712019</span></u></a></i> </p>
-
<div class="MsoNormal"
+
<u1:p></u1:p>
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
align="center"><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<p class="MsoNormal" style=""><a name="LacI"></a><b>LacI</b>: An
-
<hr align="center" size="2" width="100%"></span></div>
+
inducible promoter that was found in the part
-
<p class="MsoNormal" style="line-height: normal;"><a name="LacI"></a><b><span
+
registry.<br>
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">LacI</span></b><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;;">One
+
-
inducible Promoter which was found in the
+
-
part registry.<br>
+
<i>For more information go to:<a
<i>For more information go to:<a
-
href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010"><span
+
href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010"><u><span
-
style="color: blue;"> BBa_R0010</span></a><br style="">
+
style="color: blue;"> BBa_R0010</span></u></a><br style="">
-
<!--[endif]--></i><o:p></o:p></span></p>
+
<!--[endif]--></i></p>
-
<div class="MsoNormal"
+
<u1:p></u1:p>
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
align="center"><span
+
<hr align="center" size="2" width="100%"></div>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
<p class="MsoNormal" style=""><a name="His"></a><b>6-His Tag</b>: The
-
<hr align="center" size="2" width="100%"></span></div>
+
-
<p class="MsoNormal" style="line-height: normal;"><a name="His"></a><b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">6-His
+
-
Tag</span></b><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;;">The
+
6-Histidine Tag serves as a tag for Western
6-Histidine Tag serves as a tag for Western
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. <br>
-
like. <br>
+
<i>Note: We are using this tag as an additional method for assay beside
-
<i>Note: We are using this tag, just in case if the GFP or Luciferase
+
fluorescence of GFP and Luciferase.</i> </p>
-
does not
+
<div class="MsoNormal" style="text-align: center;" align="center">
-
work under a plate reader.</i> <o:p></o:p></span></p>
+
<hr align="center" size="2" width="100%"></div>
-
<div class="MsoNormal"
+
<br>
-
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"
+
more information go to: <a
-
align="center"><span
+
href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&amp;group=UC_Davis"><i><u><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">
+
style="color: blue;">UCDAVIS_Parts</span></u></i></a> <u1:p></u1:p>
-
<hr align="center" size="2" width="100%"></span></div>
+
-
<p class="MsoNormal" style="line-height: normal;"><a
+
-
name="ChvI_promoter"></a><b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">ChvI
+
-
promoter</span></b><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;;">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.<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>
+
-
<p class="MsoNormal" style="line-height: normal;"><a name="katA"></a><b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">KatA
+
-
promoter</span></b><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
+
-
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 <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.<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>
+
-
<p class="MsoNormal" style="line-height: normal;"><a name="aopB"></a><b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">AopB
+
-
promoter</span></b><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
+
-
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.<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>
+
-
<p class="MsoNormal" style="line-height: normal;"><a name="PhoA"></a><b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">PhoA
+
-
promoter</span></b><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;;">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.<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>
+
-
<p class="MsoNormal" style="line-height: normal;"><a name="impA"></a><b><span
+
-
style="font-size: 13pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">ImpA
+
-
promoter:</span></b><span
+
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">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. <o:p></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