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="# | + | 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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- | href="# | + | 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="border: 0px solid ; width: 87px; height: 49px;"></a><a | style="border: 0px solid ; width: 87px; height: 49px;"></a><a | ||
- | href="# | + | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="INPNC"></a>INPNC: |
- | style=" | + | </span></b><span style="">The |
- | name="INPNC"></a></span></b>< | + | 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>. </i>INP, as well as a |
- | implicated in<i> P. syringae</i> associated pathogenesis<i>. </i>INP | + | truncated derivative |
- | + | 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>. | + | on the surface of <i>E. coli (7)</i>. 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 | ||
Line 156: | Line 157: | ||
Germany) with | Germany) with | ||
codon optimization and subsequently transferred into vector (<span | codon optimization and subsequently transferred into vector (<span | ||
- | style="background: | + | 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 |
- | + | 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. <br> | + | suffix |
+ | for this part are consistent with the <i>BBF RCF-12</i> | ||
+ | standard. <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 | ||
Line 169: | Line 172: | ||
secrete | secrete | ||
any target protein. <br> | any target protein. <br> | ||
- | <u1:p></u1:p | + | <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>< | + | 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 | |
- | <div class="MsoNormal" | + | style=""> |
- | style=" | + | |
- | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="OmpA"></a>OmpA</span></b><span |
- | + | style="">: OmpA | |
- | style=" | + | is a protein found on the outer membrane of <i>E. coli</i> (13) and |
- | + | is used | |
- | is | + | as a displaying fusion protein on the cell surface. This part has |
- | + | already been | |
- | the outer membrane of <i>E. coli</i> (13) | + | 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 | + | 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 | + | 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"> | + | href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"> |
- | + | BBa_K103006</a></i> | |
- | + | <o:p></o:p></span></p> | |
- | + | <u2:p></u2:p> | |
- | + | <div class="MsoNormal" style="text-align: center;" align="center"><span | |
- | + | style=""> | |
- | + | ||
- | <div class="MsoNormal" | + | |
- | style=" | + | |
- | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="RBS"></a>RBS</span></b><span |
- | + | style="">: Ribosome | |
- | style=" | + | Binding site number 32 (BBa_J61132) from the registry is being used in |
- | + | our | |
- | + | 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. < | + | href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i>BBa_J61132</i></a><o:p></o:p></span></p> |
- | <i | + | <u2:p></u2:p> |
- | + | <div class="MsoNormal" style="text-align: center;" align="center"><span | |
- | more | + | style=""> |
- | information go to: | + | |
- | + | ||
- | href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132">< | + | |
- | + | ||
- | + | ||
- | + | ||
- | <div class="MsoNormal" | + | |
- | style=" | + | |
- | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="Terminator"></a>Terminator</span></b><span |
- | + | style="">: We are | |
- | style=" | + | using BBa_B0015, a double terminator, as our terminator in both our |
- | + | 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.< | + | <i>For more information go to:</i> <a |
- | <i | + | href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i>BBa_B0015</i></a> |
- | + | <o:p></o:p></span></p> | |
- | more | + | <u2:p></u2:p> |
- | information go to: | + | <div class="MsoNormal" style="text-align: center;" align="center"><span |
- | + | style=""> | |
- | href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015">< | + | |
- | + | ||
- | + | ||
- | + | ||
- | <div class="MsoNormal" | + | |
- | style=" | + | |
- | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="GFP"></a>GFP</span></b><span |
- | + | style="">: We are | |
- | style=" | + | using Green Fluorescent Protein as a reporter that also serves as a |
- | + | 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> </i><u2:p></u2:p><o:p></o:p></span></p> |
- | < | + | <div class="MsoNormal" style="text-align: center;" align="center"><span |
- | + | style=""> | |
- | href="http://partsregistry.org/wiki/index.php/Part:BBa_K265003">BBa_K265003</a>< | + | |
- | + | ||
- | <div class="MsoNormal" | + | |
- | style=" | + | |
- | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="Luciferase"></a>Luciferase</span></b><span |
- | + | style="">: Luciferase | |
- | style=" | + | is a firefly protein that also fluoresces, so it serves as a reporter |
- | + | 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> |
- | < | + | <u2:p></u2:p><o:p></o:p></span></p> |
- | href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019">BBa_1712019</a></ | + | <div class="MsoNormal" style="text-align: center;" align="center"><span |
- | <o:p></o:p></span></p> | + | style=""> |
- | <div class="MsoNormal" | + | |
- | style=" | + | |
- | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="LacI"></a>LacI</span></b><span |
- | + | style="">: This is an | |
- | style=" | + | inducible promoter which was found in the part registry.<br> |
- | + | <i>For more information go to:<a | |
- | inducible | + | |
- | part registry.<br> | + | |
- | + | ||
- | + | ||
- | + | ||
- | 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=""> |
- | + | <!--[endif]--></i><o:p></o:p></span></p> | |
- | style=" | + | <u2:p></u2:p> |
- | + | <div class="MsoNormal" style="text-align: center;" align="center"><span | |
- | + | style=""> | |
- | <div class="MsoNormal" | + | |
- | style=" | + | |
- | 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=" | + | <p class="MsoNormal" style=""><b><span style=""><a name="SS"></a>SS</span></b><span |
- | + | style="">: The | |
- | style=" | + | |
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). In the | lemoignei</i>, a polyhydroxybutyrate depolymerase (15). In the | ||
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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 | + | 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 | |
- | style="color: rgb(0, 41, 57);"> </span></i>The | + | would consist of a |
- | proposed constructs would | + | 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. <br> |
- | for secretion. <br> | + | <i><u1:p></u1:p>Since we expect that this part will be used in the |
- | <u1:p></u1:p | + | |
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>.< | + | style="color: rgb(0, 0, 153);">BBa_K265002</span></i></a>. <o:p></o:p></span></p> |
- | + | <u2:p></u2:p> | |
- | + | <div class="MsoNormal" style="text-align: center;" align="center"><span | |
- | + | style=""> | |
- | + | ||
- | <div class="MsoNormal" | + | |
- | style=" | + | |
- | 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"><b><span style=""><a name="Tag"></a>6-His Tag</span></b><span |
- | style=" | + | 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=" | + | highly as we |
- | + | would like. <u2:p></u2:p><br> | |
- | 6-Histidine Tag serves as a tag for | + | <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. < | + | |
- | + | ||
- | + | ||
- | We are | + | |
- | using this tag | + | |
- | under a | + | |
- | plate reader.</ | + | |
<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
Secretion Model 1:
Click on an individual part for more information.
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 pathogenesis. INP, 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).
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).
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.
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.