Team:UC Davis/Adding secretion/model 1
From 2009.igem.org
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- | href="https://2009.igem.org/Team:UC_Davis | + | href="https://2009.igem.org/Team:UC_Davis"><img alt="" |
src="https://static.igem.org/mediawiki/2009/a/a4/UCDAVIS_PIC3.png" | src="https://static.igem.org/mediawiki/2009/a/a4/UCDAVIS_PIC3.png" | ||
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- | href="https://2009.igem.org/Team:UC_Davis/ | + | href="https://2009.igem.org/Team:UC_Davis/About_Us"><b |
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- | href="https://2009.igem.org/Team:UC_Davis/ | + | href="https://2009.igem.org/Team:UC_Davis/Project"><img alt="" |
src="https://static.igem.org/mediawiki/2009/b/b9/UCDAVIS_PIC8.png" | src="https://static.igem.org/mediawiki/2009/b/b9/UCDAVIS_PIC8.png" | ||
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- | href="https://2009.igem.org/Team:UC_Davis/ | + | href="https://2009.igem.org/Team:UC_Davis/Notebook"><img alt="" |
src="https://static.igem.org/mediawiki/2009/2/2f/UCDAVIS_PIC5.png" | src="https://static.igem.org/mediawiki/2009/2/2f/UCDAVIS_PIC5.png" | ||
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- | href="https://2009.igem.org/Team:UC_Davis/ | + | href="https://2009.igem.org/Team:UC_Davis/Parts"><img alt="" |
src="https://static.igem.org/mediawiki/2009/a/a6/UCDAVIS_PIC6.png" | src="https://static.igem.org/mediawiki/2009/a/a6/UCDAVIS_PIC6.png" | ||
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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="" |
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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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<hr style="width: 100%; height: 2px;"> | <hr style="width: 100%; height: 2px;"> | ||
- | <p class="MsoNormal"><b><span | + | <p class="MsoNormal" style=""><b><span style=""><a name="INPNC"></a>INPNC: |
- | style=" | + | </span></b><span style="">The |
- | name="INPNC"></a></span></ | + | ice-nucleation protein (INP) from <i>Pseudomonas syringae</i> is used |
- | style=" | + | by its |
- | + | natural host to nucleate ice formation and is implicated in<i> | |
- | + | P.syringae</i>-associated pathogenesis<i>. </i>INP, as well as a | |
- | + | truncated derivative | |
- | + | lacking | |
- | + | the central domain (INPNC), have been used extensively for displaying | |
- | + | proteins | |
- | surface of <i>E.coli</i>( | + | on the surface of <i>E. coli (7)</i>. For instance, AldO and |
- | < | + | PhaZ1 have |
- | <p | + | been successfully displayed on the surface of <i>E. coli </i>using |
- | style=" | + | INPNC (7, |
- | + | 15). <br> | |
- | + | <u1:p></u1:p>Park <i>et al.</i> have shown that INPNC, when fused to | |
- | + | the <i>phaZ1</i> | |
- | + | gene, including its signal sequence, can serve as a suitable surface | |
- | style=" | + | delivery |
- | + | and secretion device of the otherwise toxic <i>phaZ1</i> gene product<i> | |
- | + | </i>(15). | |
- | a | + | <u1:p></u1:p>This part was synthesized by Mr. Gene (Regensburg, |
- | + | Germany) with | |
- | style=" | + | codon optimization and subsequently transferred into vector (<span |
+ | 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 | ||
+ | href="http://partsregistry.org/Part:pSB1AK3">pSB1AK3</a>).</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. <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. <br> | ||
+ | <i><u1:p></u1:p>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 </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 | <div class="MsoNormal" style="text-align: center;" align="center"><span | ||
- | style=" | + | style=""> |
<hr align="center" size="2" width="100%"></span></div> | <hr align="center" size="2" width="100%"></span></div> | ||
- | <p class="MsoNormal" style=""><a name="OmpA"></a | + | <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 | |
- | (13) | + | 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 | |
- | has | + | component |
- | already been documented on the parts registry; however, it has not been | + | of a secretion system (via fusion with a target protein linked with a |
- | tested | + | cleavable |
- | via fusion with a target protein linked with a cleavable | + | signal sequence). <i><br> |
- | sequence.<u1:p></u1:p | + | <u1:p></u1:p>We have modified this protein to be consistent with the |
- | + | BBF | |
- | + | RFC-12 Standard.<br> | |
- | + | Note: “It has remained essentially unknown how proteins of E. coli | |
- | have modified this protein to | + | outer |
- | Standard. < | + | membrane are sorted and incorporated into this membrane” (10)</i> <i><br> |
- | + | For more information go to:<a | |
- | + | href="http://partsregistry.org/wiki/index.php/Part:BBa_K103006"> | |
- | + | BBa_K103006</a></i> | |
- | + | <o:p></o:p></span></p> | |
- | + | <u2:p></u2:p> | |
- | remained essentially unknown how proteins of | + | |
- | membrane | + | |
- | are sorted and incorporated into this membrane” (10)< | + | |
- | < | + | |
- | href="http://partsregistry.org/wiki/index.php | + | |
- | + | ||
- | + | ||
<div class="MsoNormal" style="text-align: center;" align="center"><span | <div class="MsoNormal" style="text-align: center;" align="center"><span | ||
- | style=" | + | 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=" | + | style="">: Ribosome |
- | style=" | + | Binding site number 32 (BBa_J61132) from the registry is being used in |
- | + | our | |
- | being used in our secretion system.< | + | secretion system. <u2:p></u2:p><br> |
- | + | <i>For more information go to:</i> <a | |
- | + | href="http://partsregistry.org/wiki/index.php/Part:BBa_J61132"><i>BBa_J61132</i></a><o:p></o:p></span></p> | |
- | + | <u2:p></u2:p> | |
- | more information go to: | + | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<div class="MsoNormal" style="text-align: center;" align="center"><span | <div class="MsoNormal" style="text-align: center;" align="center"><span | ||
- | style=" | + | 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=" | + | style="">: We are |
- | style=" | + | using BBa_B0015, a double terminator, as our terminator in both our |
- | We are using BBa_B0015, a double terminator, as our terminator in | + | secretion |
- | both our secretion and pH system.< | + | and pH system.<u2:p></u2:p><br> |
- | + | <i>For more information go to:</i> <a | |
- | + | href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015"><i>BBa_B0015</i></a> | |
- | + | <o:p></o:p></span></p> | |
- | more information go to: | + | <u2:p></u2:p> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<div class="MsoNormal" style="text-align: center;" align="center"><span | <div class="MsoNormal" style="text-align: center;" align="center"><span | ||
- | style=" | + | 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=" | + | style="">: We are |
- | style=" | + | using Green Fluorescent Protein as a reporter that also serves as a |
- | We are using Green Fluorescent Protein as a reporter that also | + | small |
- | serves as a small protein in testing our secretion system.</ | + | protein in testing our secretion system.<br> |
- | + | <i>For more informaiton go to: </i><a | |
+ | 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 | <div class="MsoNormal" style="text-align: center;" align="center"><span | ||
- | style=" | + | 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=" | + | style="">: Luciferase |
- | style=" | + | is a firefly protein that also fluoresces, so it serves as a reporter |
- | + | as well | |
- | serves as a reporter as well as a testable large protein.< | + | as a testable large protein.<br> |
- | + | <i>For more information go to: <a | |
- | + | href="http://partsregistry.org/wiki/index.php/Part:BBa_I712019">BBa_1712019</a></i> | |
+ | <u2:p></u2:p><o:p></o:p></span></p> | ||
<div class="MsoNormal" style="text-align: center;" align="center"><span | <div class="MsoNormal" style="text-align: center;" align="center"><span | ||
- | style=" | + | style=""> |
<hr align="center" size="2" width="100%"></span></div> | <hr align="center" size="2" width="100%"></span></div> | ||
- | <p class="MsoNormal" style=""><a name="LacI"></a | + | <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 | |
- | + | href="http://partsregistry.org/wiki/index.php/Part:BBa_R0010"> | |
- | href="http://partsregistry.org/wiki/index.php | + | BBa_R0010</a><br style=""> |
- | style=""> | + | <!--[endif]--></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="text-align: center;" align="center"><span | ||
- | style=" | + | style=""> |
<hr align="center" size="2" width="100%"></span></div> | <hr align="center" size="2" width="100%"></span></div> | ||
- | <p class="MsoNormal"><a name="SS"></a><b><span | + | <p class="MsoNormal" style=""><b><span style=""><a name="SS"></a>SS</span></b><span |
- | style=" | + | style="">: The |
- | style=" | + | signal sequence (SS) for the <i>phaZ1 </i>gene product of <i>Paucimonas |
- | + | lemoignei</i>, a polyhydroxybutyrate depolymerase (15). In the | |
- | + | native | |
- | + | protein the signal sequence is cleaved between residues Ala37 and | |
- | style=" | + | Leu38. |
- | + | Park <i>et al. </i>have showed that the fusion of the complete <i>phaZ1 | |
- | style=" | + | </i>gene |
- | have modified this protein to | + | (including SS) and a truncated ice nucleation protein from <i>Pseudomonas |
- | Standard.</ | + | syringae</i> (<a |
- | style=" | + | 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. <br> | ||
+ | <u1:p></u1:p>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 (<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 consist of a | ||
+ | membrane anchor (INPNC or OmpA) followed by the cleavable signal | ||
+ | sequence and | ||
+ | finally a target protein marked for secretion. <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 the | ||
+ | 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> | ||
+ | <u2:p></u2:p> | ||
<div class="MsoNormal" style="text-align: center;" align="center"><span | <div class="MsoNormal" style="text-align: center;" align="center"><span | ||
- | style="font-family: "Times New Roman","serif";"> | + | style=""> |
+ | <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="">: The 6-Histidine Tag serves as a tag for | ||
+ | Western Blotting if our fluorescent reporters are not expressed as | ||
+ | highly as we | ||
+ | would like. <u2:p></u2:p><br> | ||
+ | <i>Note: We are using this tag just in case the GFP or Luciferase | ||
+ | do not | ||
+ | work under a plate reader.</i> <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: "Times New Roman","serif";"> | ||
<hr align="center" size="2" width="100%"></span></div> | <hr align="center" size="2" width="100%"></span></div> | ||
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<b><i><span | <b><i><span | ||
style="font-size: 12pt; line-height: 115%; font-family: "Times New Roman","serif";"></span></i></b><big><big><big><span | style="font-size: 12pt; line-height: 115%; font-family: "Times New Roman","serif";"></span></i></b><big><big><big><span |
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.