Team:UC Davis/Adding secretion

From 2009.igem.org

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<p class="MsoNormal" style="text-align: center;" align="center"><span
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<p class="MsoNormal" style="text-align: left;"><big><span style="">&nbsp;&nbsp;&nbsp;
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style="font-size: 13.5pt;">The purpose of the secretion system is to
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&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <span
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introduce a
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style="font-family: Times New Roman,Times,serif;">The purpose of the
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method of secreting target proteins we wish to synthesize in the
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secretion system is to
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marvelous
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introduce a method of secreting target proteins we wish to synthesize
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host: E.coli. For our secretion system </span><span
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in the
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style="font-size: 13.5pt;">we have taken the idea from (<a
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marvelous host: E.coli. For our secretion system we have taken the idea
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href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).
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from (</span><a style="font-family: Times New Roman,Times,serif;"
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Park <i>et. al</i>
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href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a><span
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showed that construct containing the truncated from of ice nucleation
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style="font-family: Times New Roman,Times,serif;">). Park </span><i
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protein
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style="font-family: Times New Roman,Times,serif;">et.
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(INPNC) where found to complete coding region of phaZ1, including its
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al</i><span style="font-family: Times New Roman,Times,serif;"> showed
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that construct containing the truncated from of ice nucleation
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protein (INPNC) where found to 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
signal
signal
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sequence could lead to stable secretion of the enzyme (<a
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sequence serve as carries for a target protein.</span><o:p
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href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).
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style="font-family: Times New Roman,Times,serif;"></o:p></span></big></p>
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We have decided to
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<p class="MsoNormal" style="font-family: Times New Roman,Times,serif;"><big><span
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see if we could co-opt this system to make a more generalized inducible
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style=""><br>
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system
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<big><b>Possible challenges</b>:</big><o:p></o:p></span></big></p>
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for protein secretion in which the INPNC and phaZ1 signal sequence
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<p class="MsoNormal"
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serve as
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style="margin-left: 30pt; text-align: left; font-family: Times New Roman,Times,serif;"><big><span
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carries for a target protein.</span></p>
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style="">1. Efficient recognition of the change site may
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<p class="MsoNormal"><span style="font-size: 13.5pt;"><br>
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<span
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<b>Possible challenges</b>:</span></p>
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<div style="text-align: left;">
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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></span>
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</div>
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some of the phaZ1 protein we have only cloned the
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<p style="text-align: left; margin-left: 40px;" class="MsoNormal"><span
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signal sequence up to the cleavage site.<br>
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style="font-size: 13.5pt;">1. Efficient recognition of
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2. The system may only work for protein in a narrow site range.<br>
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the change site may <span
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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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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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<p class="MsoNormal"
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some of the phaZ1 protein we have only cloned the signal sequence up to
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style="text-align: center; font-family: Times New Roman,Times,serif;"
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the
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align="center"><big><b><span style=""><br>
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cleavage site.<br>
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<big>Advantages:</big></span></b><span style=""><o:p></o:p></span></big></p>
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2. The system may only work
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<ol style="text-align: left;" start="1" type="1">
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for protein in a narrow site range.<br>
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<li style="font-family: Times New Roman,Times,serif;"
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3. The expression levels of the system may be low.</span></p>
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class="MsoNormal"><big><span style=""><u1:p></u1:p>&nbsp; In our
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<p class="MsoNormal" style="text-align: center;" align="center"><b><br>
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secretion system, we are using two genes with different sizes as our
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<big>Advantages:<br>
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target secretion genes, GFP being short in length and Luciferase being
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</big></b></p>
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comparably long, would test our secretion system and its ability to
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<u1:p></u1:p>
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secrete small and large proteins. <o:p></o:p></span></big></li>
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<div style="text-align: left;"></div>
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<li style="font-family: Times New Roman,Times,serif;"
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<ol style="text-align: left;">
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class="MsoNormal"><big><span style="">&nbsp; Testing multiple
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<li><span style="font-size: 13.5pt;">&nbsp; In our secretion system,
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different combinations, Signal Sequence plus ompA/INPNC would help us
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we are using two genes with
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find the best combination for our secretion system based on their
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different sizes as our target secretion genes, GFP being short in
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secretion ability and rank them from strongest to weakest in strength
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length and
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(in this specific system).<o:p></o:p></span></big></li>
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Luciferase being comparably long, would test our secretion system and
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<li class="MsoNormal"><span style=""><big
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its
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style="font-family: Times New Roman,Times,serif;">&nbsp; Also, we have
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ability to secrete small and large proteins. </span></li>
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submitted the BioBricks to the parts registry; therefore they can be
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<li><span style="font-size: 13.5pt;">&nbsp; Testing multiple
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used in future studies (for different purposes).</big><o:p></o:p></span></li>
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different combinations,
+
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Signal Sequence plus ompA/INPNC would help us find the best combination
+
-
for our
+
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secretion system based on their secretion ability and rank them from
+
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strongest
+
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to weakest in strength (in this specific system).</span></li>
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<li><span style="font-size: 13.5pt;">&nbsp; Also, we have submitted
+
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the BioBricks to the parts regisitry, therefore they can be used in
+
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future studies (for different purposes).</span></li>
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</ol>
</ol>
<br>
<br>

Revision as of 16:20, 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 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




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