Team:UC Davis/Adding secretion

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<p class="MsoNormal" style="text-align: left;"><big><span style="">&nbsp;&nbsp;&nbsp;
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&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <span
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style="font-family: Times New Roman,Times,serif;">The purpose of the
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secretion system is to
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introduce a method of secreting target proteins we wish to synthesize
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in the
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marvelous host: <i>E.coli</i>. Park </span><i
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style="font-family: Times New Roman,Times,serif;">et.
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al</i><span style="font-family: Times New Roman,Times,serif;"> showed
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that the truncated form of ice nucleation
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protein (INPNC) containing the complete coding region of phaZ1,
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including its
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signal sequence, could lead to stable secretion of the enzyme (</span><a
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style="font-family: Times New Roman,Times,serif;"
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href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a><span
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style="font-family: Times New Roman,Times,serif;">). We have
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decided to see if we could co-opt this system to make a more
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generalized
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inducible system for protein secretion in which the INPNC and phaZ1
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signal
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sequence serve as carriers for a target protein.</span><o:p
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style="font-family: Times New Roman,Times,serif;"></o:p></span></big></p>
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<p class="MsoNormal" style="font-family: Times New Roman,Times,serif;"><big><span
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style=""><br>
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<big><b>Possible challenges</b>:</big><o:p></o:p></span></big></p>
<p class="MsoNormal"
<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="margin-left: 30pt; text-align: left; font-family: Times New Roman,Times,serif;"><big><span
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">Advantages
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style="">1. Efficient recognition of the cleavage site may require<span
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of having several models:</span></b><b><span
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<div style="text-align: left;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span
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some of the non-signal sequence portion of the phaZ1 protein (we have
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style="font-family: Times New Roman,Times,serif;">&nbsp;&nbsp;&nbsp;&nbsp;
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only cloned the
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<big>The purpose of the secretion system is to introduce a method
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signal sequence up to the cleavage site).<br>
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of excreting target proteins we wish to synthesize in the marvelous
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2. The system may only work for proteins in a narrow site range.<br>
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host: E.coli
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3. The expression levels of the system may be low.<o:p></o:p></span></big></p>
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HB101.&nbsp; Through this approach of being able to secrete specific
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<p class="MsoNormal"
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target
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style="text-align: center; font-family: Times New Roman,Times,serif;"
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proteins, we hope to serve as a chassis to the rest of the biological
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align="center"><big><b><span style=""><br>
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world.</big></span><big><br
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<big>Advantages:</big></span></b><span style=""><o:p></o:p></span></big></p>
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<ol style="text-align: left;" start="1" type="1">
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<span style="font-family: Times New Roman,Times,serif;">&nbsp;&nbsp;&nbsp;
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<li style="font-family: Times New Roman,Times,serif;"
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&nbsp;&nbsp;&nbsp; In our secretion system, we are using two
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class="MsoNormal"><big><span style=""><u1:p></u1:p>&nbsp; In our
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genes with different sizes as our target secretion genes, GFP being
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secretion system, we are using two genes with different sizes as our
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short in
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target secretion genes; GFP being short in length and Luciferase being
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length and Luciferase being comparably long would test our secretion
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comparably long, these would test the ability of our secretion system
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system and
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to secrete proteins of different sizes. <o:p></o:p></span></big></li>
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its ability to secrete small and large proteins. </span><br
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<li style="font-family: Times New Roman,Times,serif;"
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style="font-family: Times New Roman,Times,serif;">
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class="MsoNormal"><big><span style="">&nbsp; Testing various
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<span style="font-family: Times New Roman,Times,serif;">&nbsp;&nbsp;&nbsp;
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combinations of the Signal Sequence with either ompA or INPNC would
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&nbsp;&nbsp;&nbsp; Testing multiple different
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help us
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combinations; Signal Sequence alone, Signal Sequence plus ompA/INPNC
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find the best combination for our secretion system based on their
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and
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secretion ability and allow us to rank the combinations from strongest
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ompA/INPNC alone, would help us find the best combination for our
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to weakest in strength.
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secretion
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(in this specific system).<o:p></o:p></span></big></li>
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system based on their secretion ability and rank them from strongest to
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<li class="MsoNormal"><span style=""><big
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weakest
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style="font-family: Times New Roman,Times,serif;">&nbsp; We have
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in strength (in this specific system).</span></big></div>
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submitted the BioBricks to the parts registry; therefore they can be
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used in future studies (for different purposes).</big><o:p></o:p></span></li>
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</ol>
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<br>
<div style="text-align: left;"><big><big
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href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_2"><img
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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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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

Latest revision as of 02:35, 22 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. Park et. al showed that the truncated form of ice nucleation protein (INPNC) containing the 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 carriers for a target protein.


Possible challenges:

1. Efficient recognition of the cleavage site may require some of the non-signal sequence portion of the phaZ1 protein (we have only cloned the signal sequence up to the cleavage site).
2. The system may only work for proteins 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, these would test the ability of our secretion system to secrete proteins of different sizes.
  2.   Testing various combinations of the Signal Sequence with either ompA or INPNC would help us find the best combination for our secretion system based on their secretion ability and allow us to rank the combinations from strongest to weakest in strength. (in this specific system).
  3.   We have submitted the BioBricks to the parts registry; 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