Team:UC Davis/Adding secretion

From 2009.igem.org

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</span></small></span><small><small><span
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style="text-decoration: underline;"><br>
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</span></small></small></big></big></big>
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<p class="MsoNormal" style="text-align: center;" align="center"><span
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style="font-size: 13.5pt;">The purpose of the secretion system is to
 +
introduce a
 +
method of secreting target proteins we wish to synthesize in the
 +
marvelous
 +
host: E.coli. For our secretion system </span><span
 +
style="font-size: 13.5pt;">we have taken the idea from (<a
 +
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).
 +
Park <i>et. al</i>
 +
showed that construct containing the truncated from of ice nucleation
 +
protein
 +
(INPNC) where found to complete coding region of phaZ1, including its
 +
signal
 +
sequence could lead to stable secretion of the enzyme (<a
 +
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).
 +
We have decided to
 +
see if we could co-opt this system to make a more generalized inducible
 +
system
 +
for protein secretion in which the INPNC and phaZ1 signal sequence
 +
serve as
 +
carries for a target protein.</span></p>
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<p class="MsoNormal"><span style="font-size: 13.5pt;"><br>
 +
<b>Possible challenges</b>:</span></p>
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<div style="text-align: left;">
 +
</div>
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<p style="text-align: left; margin-left: 40px;" class="MsoNormal"><span
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style="font-size: 13.5pt;">1. Efficient recognition of
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the change site may <span
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style="background: yellow none repeat scroll 0%; -moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial;">()</span>
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some of the phaZ1 protein we have only cloned the signal sequence up to
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the
 +
cleavage site.<br>
 +
2. The system may only work
 +
for protein in a narrow site range.<br>
 +
3. The expression levels of the system may be low.</span></p>
 +
<p class="MsoNormal" style="text-align: center;" align="center"><b><br>
 +
<big>Advantages:<br>
 +
</big></b></p>
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<u1:p></u1:p>
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<div style="text-align: left;"></div>
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<ol style="text-align: left;">
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<li><span style="font-size: 13.5pt;">&nbsp; In our secretion system,
 +
we are using two genes with
 +
different sizes as our target secretion genes, GFP being short in
 +
length and
 +
Luciferase being comparably long, would test our secretion system and
 +
its
 +
ability to secrete small and large proteins. </span></li>
 +
<li><span style="font-size: 13.5pt;">&nbsp; Testing multiple
 +
different combinations,
 +
Signal Sequence plus ompA/INPNC would help us find the best combination
 +
for our
 +
secretion system based on their secretion ability and rank them from
 +
strongest
 +
to weakest in strength (in this specific system).</span></li>
 +
<li><span style="font-size: 13.5pt;">&nbsp; Also, we have submitted
 +
the BioBricks to the parts regisitry, therefore they can be used in
 +
future studies (for different purposes).</span></li>
 +
</ol>
<br>
<br>
-
</span></small></span></big></big></big>
 
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<p class="MsoNormal"
 
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style="margin-bottom: 0.0001pt; line-height: normal; text-align: center;"><b><span
 
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Advantages
 
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of having several models:</span></b><b><span
 
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><o:p></o:p></span></b></p>
 
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<div style="text-align: left;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span
 
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style="font-family: Times New Roman,Times,serif;">&nbsp;&nbsp;&nbsp;&nbsp;
 
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<big>The purpose of the secretion system is to introduce a method
 
-
of excreting target proteins we wish to synthesize in the marvelous
 
-
host: E.coli
 
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HB101.&nbsp; Through this approach of being able to secrete specific
 
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target
 
-
proteins, we hope to serve as a chassis to the rest of the biological
 
-
world.</big></span><big><br
 
-
style="font-family: Times New Roman,Times,serif;">
 
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<span style="font-family: Times New Roman,Times,serif;">&nbsp;&nbsp;&nbsp;
 
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&nbsp;&nbsp;&nbsp; In our secretion system, we are using two
 
-
genes with different sizes as our target secretion genes, GFP being
 
-
short in
 
-
length and Luciferase being comparably long would test our secretion
 
-
system and
 
-
its ability to secrete small and large proteins. </span><br
 
-
style="font-family: Times New Roman,Times,serif;">
 
-
<span style="font-family: Times New Roman,Times,serif;">&nbsp;&nbsp;&nbsp;
 
-
&nbsp;&nbsp;&nbsp; Testing multiple different
 
-
combinations; Signal Sequence alone, Signal Sequence plus ompA/INPNC
 
-
and
 
-
ompA/INPNC alone, would help us find the best combination for our
 
-
secretion
 
-
system based on their secretion ability and rank them from strongest to
 
-
weakest
 
-
in strength (in this specific system).</span></big></div>
 
<div style="text-align: left;"><big><big
<div style="text-align: left;"><big><big
style="font-family: Times New Roman,Times,serif;"><big><big><span
style="font-family: Times New Roman,Times,serif;"><big><big><span
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style="border: 0px solid ; width: 95px; height: 63px;"></a><a
style="border: 0px solid ; width: 95px; height: 63px;"></a><a
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href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_2"><img
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href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_4"><img
alt="" src="https://static.igem.org/mediawiki/2009/a/a4/UCDAVIS_PIC13.png"
alt="" src="https://static.igem.org/mediawiki/2009/a/a4/UCDAVIS_PIC13.png"
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style="border: 0px solid ; width: 95px; height: 63px;"></a><a
 
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href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_3"><img
 
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alt="" src="https://static.igem.org/mediawiki/2009/9/9a/UCDAVIS_PIC14.png"
 
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style="border: 0px solid ; width: 95px; height: 63px;"></a><a
 
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href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_4"><img
 
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alt="" src="https://static.igem.org/mediawiki/2009/b/ba/UCDAVIS_PIC15.png"
 
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style="border: 0px solid ; width: 95px; height: 63px;"></a><a
 
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href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_5"><img
 
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alt="" src="https://static.igem.org/mediawiki/2009/5/5d/UCDAVIS_PIC16.png"
 
style="border: 0px solid ; width: 95px; height: 63px;"></a>&nbsp;&nbsp;&nbsp;
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</span><span style="font-style: italic;"><big><span
</span><span style="font-style: italic;"><big><span

Revision as of 16:15, 21 October 2009

Adding Secretion

 
Adding Secretion:
General model for secretion     Secretion Models     Why test different genes

General model for secretion system:

The purpose of the secretion system is to introduce a method of secreting target proteins we wish to synthesize in the marvelous host: E.coli. For our secretion system we have taken the idea from (15). Park et. al showed that construct containing the truncated from of ice nucleation protein (INPNC) where found to complete coding region of phaZ1, including its signal sequence could lead to stable secretion of the enzyme (15). We have decided to see if we could co-opt this system to make a more generalized inducible system for protein secretion in which the INPNC and phaZ1 signal sequence serve as carries for a target protein.


Possible challenges:

1. Efficient recognition of the change site may () some of the phaZ1 protein we have only cloned the signal sequence up to the cleavage site.
2. The system may only work for protein in a narrow site range.
3. The expression levels of the system may be low.


Advantages:

  1.   In our secretion system, we are using two genes with different sizes as our target secretion genes, GFP being short in length and Luciferase being comparably long, would test our secretion system and its ability to secrete small and large proteins.
  2.   Testing multiple different combinations, Signal Sequence plus ompA/INPNC would help us find the best combination for our secretion system based on their secretion ability and rank them from strongest to weakest in strength (in this specific system).
  3.   Also, we have submitted the BioBricks to the parts regisitry, therefore they can be used in future studies (for different purposes).


Secretion Models:
Click on a specific model for more information:
   

Why test different genes in our secretion system?

     There is wide range of genes present in our environment. Therefore, it is important to measure our secretion system’s ability to secrete genes of various sizes.

Molecular data on some proteins

Name
 Molecular Weight (kD)
Myosin
 200.0
-Galactosidase
 116.3
Phosphorylase b
 97.4
Ovalbumin
 45.0
Carbonic anhydrase
 31.0
Aprotinin
 6.5
Insulin, B chain, oxidized
 3.5




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