Team:UC Davis/Adding secretion

From 2009.igem.org

(Difference between revisions)
 
(3 intermediate revisions not shown)
Line 83: Line 83:
style="text-decoration: underline;"><br>
style="text-decoration: underline;"><br>
</span></small></small></big></big></big>
</span></small></small></big></big></big>
-
<p class="MsoNormal" style="text-align: center;" align="center"><span
+
<p class="MsoNormal" style="text-align: left;"><big><span style="">&nbsp;&nbsp;&nbsp;
-
style="font-size: 13.5pt;">The purpose of the secretion system is to
+
&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; <span
-
introduce a
+
style="font-family: Times New Roman,Times,serif;">The purpose of the
-
method of secreting target proteins we wish to synthesize in the
+
secretion system is to
-
marvelous
+
introduce a method of secreting target proteins we wish to synthesize
-
host: E.coli. For our secretion system </span><span
+
in the
-
style="font-size: 13.5pt;">we have taken the idea from (<a
+
marvelous host: <i>E.coli</i>. Park </span><i
-
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).
+
style="font-family: Times New Roman,Times,serif;">et.
-
Park <i>et. al</i>
+
al</i><span style="font-family: Times New Roman,Times,serif;"> showed
-
showed that construct containing the truncated from of ice nucleation
+
that the truncated form of ice nucleation
-
protein
+
protein (INPNC) containing the complete coding region of phaZ1,
-
(INPNC) where found to complete coding region of phaZ1, including its
+
including its
 +
signal sequence, could lead to stable secretion of the enzyme (</span><a
 +
style="font-family: Times New Roman,Times,serif;"
 +
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a><span
 +
style="font-family: Times New Roman,Times,serif;">). 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
signal
-
sequence could lead to stable secretion of the enzyme (<a
+
sequence serve as carriers for a target protein.</span><o:p
-
href="https://2009.igem.org/Team:UC_Davis/Contacts_References">15</a>).
+
style="font-family: Times New Roman,Times,serif;"></o:p></span></big></p>
-
We have decided to
+
<p class="MsoNormal" style="font-family: Times New Roman,Times,serif;"><big><span
-
see if we could co-opt this system to make a more generalized inducible
+
style=""><br>
-
system
+
<big><b>Possible challenges</b>:</big><o:p></o:p></span></big></p>
-
for protein secretion in which the INPNC and phaZ1 signal sequence
+
<p class="MsoNormal"
-
serve as
+
style="margin-left: 30pt; text-align: left; font-family: Times New Roman,Times,serif;"><big><span
-
carries for a target protein.</span></p>
+
style="">1. Efficient recognition of the cleavage site may require<span
-
<p class="MsoNormal"><span style="font-size: 13.5pt;"><br>
+
style="-moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial; background-attachment: scroll;"><span
-
<b>Possible challenges</b>:</span></p>
+
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>
-
<div style="text-align: left;">
+
some of the non-signal sequence portion of the phaZ1 protein (we have
-
</div>
+
only cloned the
-
<p style="text-align: left; margin-left: 40px;" class="MsoNormal"><span
+
signal sequence up to the cleavage site).<br>
-
style="font-size: 13.5pt;">1. Efficient recognition of
+
2. The system may only work for proteins in a narrow site range.<br>
-
the change site may <span
+
3. The expression levels of the system may be low.<o:p></o:p></span></big></p>
-
style="background: yellow none repeat scroll 0%; -moz-background-clip: -moz-initial; -moz-background-origin: -moz-initial; -moz-background-inline-policy: -moz-initial;">()</span>
+
<p class="MsoNormal"
-
some of the phaZ1 protein we have only cloned the signal sequence up to
+
style="text-align: center; font-family: Times New Roman,Times,serif;"
-
the
+
align="center"><big><b><span style=""><br>
-
cleavage site.<br>
+
<big>Advantages:</big></span></b><span style=""><o:p></o:p></span></big></p>
-
2. The system may only work
+
<ol style="text-align: left;" start="1" type="1">
-
for protein in a narrow site range.<br>
+
<li style="font-family: Times New Roman,Times,serif;"
-
3. The expression levels of the system may be low.</span></p>
+
class="MsoNormal"><big><span style=""><u1:p></u1:p>&nbsp; In our
-
<p class="MsoNormal" style="text-align: center;" align="center"><b><br>
+
secretion system, we are using two genes with different sizes as our
-
<big>Advantages:<br>
+
target secretion genes; GFP being short in length and Luciferase being
-
</big></b></p>
+
comparably long, these would test the ability of our secretion system
-
<u1:p></u1:p>
+
to secrete proteins of different sizes. <o:p></o:p></span></big></li>
-
<div style="text-align: left;"></div>
+
<li style="font-family: Times New Roman,Times,serif;"
-
<ol style="text-align: left;">
+
class="MsoNormal"><big><span style="">&nbsp; Testing various
-
<li><span style="font-size: 13.5pt;">&nbsp; In our secretion system,
+
combinations of the Signal Sequence with either ompA or INPNC would
-
we are using two genes with
+
help us
-
different sizes as our target secretion genes, GFP being short in
+
find the best combination for our secretion system based on their
-
length and
+
secretion ability and allow us to rank the combinations from strongest
-
Luciferase being comparably long, would test our secretion system and
+
to weakest in strength.
-
its
+
(in this specific system).<o:p></o:p></span></big></li>
-
ability to secrete small and large proteins. </span></li>
+
<li class="MsoNormal"><span style=""><big
-
<li><span style="font-size: 13.5pt;">&nbsp; Testing multiple
+
style="font-family: Times New Roman,Times,serif;">&nbsp; We have
-
different combinations,
+
submitted the BioBricks to the parts registry; therefore they can be
-
Signal Sequence plus ompA/INPNC would help us find the best combination
+
used in future studies (for different purposes).</big><o:p></o:p></span></li>
-
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>
</ol>
<br>
<br>
Line 163: Line 163:
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
-
href="https://2009.igem.org/Team:UC_Davis/Adding_secretion/model_4"><img
+
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