Team:UC Davis/Adding secretion

From 2009.igem.org

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introduce a method of secreting target proteins we wish to synthesize
introduce a method of secreting target proteins we wish to synthesize
in the
in the
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marvelous host: E.coli. For our secretion system we have taken the idea
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marvelous host: <i>E.coli</i>. Park </span><i
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from (</span><a style="font-family: Times New Roman,Times,serif;"
+
-
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;">). Park </span><i
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style="font-family: Times New Roman,Times,serif;">et.
style="font-family: Times New Roman,Times,serif;">et.
al</i><span style="font-family: Times New Roman,Times,serif;"> showed
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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that the truncated form of ice nucleation
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protein (INPNC) where found to complete coding region of phaZ1,
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protein (INPNC) containing the complete coding region of phaZ1,
including its
including its
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signal sequence could lead to stable secretion of the enzyme (</span><a
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signal sequence, could lead to stable secretion of the enzyme (</span><a
style="font-family: Times New Roman,Times,serif;"
style="font-family: Times New Roman,Times,serif;"
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a><span
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a><span
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inducible system for protein secretion in which the INPNC and phaZ1
inducible system for protein secretion in which the INPNC and phaZ1
signal
signal
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sequence serve as carries for a target protein.</span><o:p
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sequence serve as carriers for a target protein.</span><o:p
style="font-family: Times New Roman,Times,serif;"></o:p></span></big></p>
style="font-family: Times New Roman,Times,serif;"></o:p></span></big></p>
<p class="MsoNormal" style="font-family: Times New Roman,Times,serif;"><big><span
<p class="MsoNormal" style="font-family: Times New Roman,Times,serif;"><big><span
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<p class="MsoNormal"
<p class="MsoNormal"
style="margin-left: 30pt; text-align: left; font-family: Times New Roman,Times,serif;"><big><span
style="margin-left: 30pt; text-align: left; font-family: Times New Roman,Times,serif;"><big><span
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style="">1. Efficient recognition of the change site may
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style="">1. Efficient recognition of the cleavage site may require<span
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<span
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style="-moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial; background-attachment: scroll;"><span
style="-moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial; background-attachment: scroll;"><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></span>
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style="background: yellow none repeat scroll 0% 50%; -moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial;"></span></span>
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some of the phaZ1 protein we have only cloned the
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some of the non-signal sequence portion of the phaZ1 protein (we have
-
signal sequence up to the cleavage site.<br>
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only cloned the
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2. The system may only work for protein in a narrow site range.<br>
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signal sequence up to the cleavage site).<br>
 +
2. The system may only work for proteins in a narrow site range.<br>
3. The expression levels of the system may be low.<o:p></o:p></span></big></p>
3. The expression levels of the system may be low.<o:p></o:p></span></big></p>
<p class="MsoNormal"
<p class="MsoNormal"
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class="MsoNormal"><big><span style=""><u1:p></u1:p>&nbsp; In our
class="MsoNormal"><big><span style=""><u1:p></u1:p>&nbsp; In our
secretion system, we are using two genes with different sizes as our
secretion system, we are using two genes with different sizes as our
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target secretion genes, GFP being short in length and Luciferase being
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target secretion genes; GFP being short in length and Luciferase being
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comparably long, would test our secretion system and its ability to
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comparably long, these would test the ability of our secretion system
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secrete small and large proteins. <o:p></o:p></span></big></li>
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to secrete proteins of different sizes. <o:p></o:p></span></big></li>
<li style="font-family: Times New Roman,Times,serif;"
<li style="font-family: Times New Roman,Times,serif;"
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class="MsoNormal"><big><span style="">&nbsp; Testing multiple
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class="MsoNormal"><big><span style="">&nbsp; Testing various
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different combinations, Signal Sequence plus ompA/INPNC would help us
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combinations of the Signal Sequence with either ompA or INPNC would
 +
help us
find the best combination for our secretion system based on their
find the best combination for our secretion system based on their
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secretion ability and rank them from strongest to weakest in strength
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secretion ability and allow us to rank the combinations from strongest
 +
to weakest in strength.
(in this specific system).<o:p></o:p></span></big></li>
(in this specific system).<o:p></o:p></span></big></li>
<li class="MsoNormal"><span style=""><big
<li class="MsoNormal"><span style=""><big
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style="font-family: Times New Roman,Times,serif;">&nbsp; Also, we have
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style="font-family: Times New Roman,Times,serif;">&nbsp; We have
submitted the BioBricks to the parts registry; therefore they can be
submitted the BioBricks to the parts registry; therefore they can be
used in future studies (for different purposes).</big><o:p></o:p></span></li>
used in future studies (for different purposes).</big><o:p></o:p></span></li>
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alt="" src="https://static.igem.org/mediawiki/2009/b/ba/UCDAVIS_PIC12.png"
alt="" src="https://static.igem.org/mediawiki/2009/b/ba/UCDAVIS_PIC12.png"
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_4"><img
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href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_2"><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"
style="border: 0px solid ; width: 95px; height: 63px;"></a>&nbsp;&nbsp;&nbsp;
style="border: 0px solid ; width: 95px; height: 63px;"></a>&nbsp;&nbsp;&nbsp;

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




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