Difference between revisions of "Team:Aix-Marseille/Design"

Line 1: Line 1:
 
{{Aix-Marseille|title=Design|toc=__TOC__}}
 
{{Aix-Marseille|title=Design|toc=__TOC__}}
  
−
<html>
+
===Engineering M13===
 +
[[File:T--Aix-Marseille--M13K07.png|350px|right]]
  
 +
In order to engineered multiple phages to infect various pathogenes we first decided to remove D1 and D2. As we wanted to insert those two domains in the p3 of the M13 genome. Thus we use M13KO7 from New England BioLab. '''M13KO7''' is an M13 derivative which carries the mutation Met40Ile in gII , with the origin of replication from P15A and the kanamycin resistance gene from Tn903 both inserted within the M13 origin of replication.
  
 +
In '''M13KO7''' we wanted to insert two restriction site (AvrII and BspI) which are compatible with XbaI and AgeI. Thus, we create two types of biobrick, one with the signal sequence of M13, and the other one with D1 and D2 of another p3 from another filamentous phages.
  
 +
In our design we wanted to keep the signal sequence and D3 of M13, because their are crucial for the formation of the phage. We just want to insert D1 and D2 from another phages (we’ll call it X).
  
−
<div class="column full_size">
+
[[File:T--Aix-Marseille--P3map.png|700px|center]]
−
<h1>Design</h1>
+
−
<p>
+
−
Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
+
−
</p>
+
  
−
<p>
+
Another way to engineered M13, is to remove entierely the protein III from the phage genome and to reconstruct it in another plasmid. Thus, we create another part : p3_D3, which is the domain involved in the assembly and release of M13 particles.
−
This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:Aix-Marseille/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
+
−
</p>
+
  
−
</div>
+
[[Team:Aix-Marseille/M13KO7|Read more…]]
  
−
<div class="column half_size">
+
===Phagemid===
−
<h5>What should this page contain?</h5>
+
−
<ul>
+
−
<li>Explanation of the engineering principles your team used in your design</li>
+
−
<li>Discussion of the design iterations your team went through</li>
+
−
<li>Experimental plan to test your designs</li>
+
−
</ul>
+
  
−
</div>
+
As M13KO7 genome is not capable to be used for the phage assemblage, we will use a phagemid which will carry a toxin to assemble our ingeneered M13 phage.
  
−
<div class="column half_size">
+
We thought about using the Super Nova toxin (BBa_K1491017), made by the iGEM team Carnegie Mellon in 2014. This toxin has multiple benefits, because of the production of ROS occuring only in a yellow - orange light, we can produce phages-like particules carring this toxin without killing ''E.coli''. However, when this toxin will be produced in ''X. fastidiosa'' with the light coming from the sun, the bacterium will be harmed, even if it is in the Xylem vessels.
−
<h5>Inspiration</h5>
+
To optimise the production of this toxin in ''X. fastidiosa'' we tried to find a strong and constitutive promoter of this bacterium.
−
<ul>
+
−
<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
+
−
<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
+
−
<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
+
−
</ul>
+
−
</div>
+
  
 +
We thought about multiple ways to engineer our phagemid.
  
−
 
+
[[Team:Aix-Marseille/phagemid|Read more…]]
−
</html>
+

Revision as of 13:38, 31 August 2017

Design

Engineering M13

T--Aix-Marseille--M13K07.png

In order to engineered multiple phages to infect various pathogenes we first decided to remove D1 and D2. As we wanted to insert those two domains in the p3 of the M13 genome. Thus we use M13KO7 from New England BioLab. M13KO7 is an M13 derivative which carries the mutation Met40Ile in gII , with the origin of replication from P15A and the kanamycin resistance gene from Tn903 both inserted within the M13 origin of replication.

In M13KO7 we wanted to insert two restriction site (AvrII and BspI) which are compatible with XbaI and AgeI. Thus, we create two types of biobrick, one with the signal sequence of M13, and the other one with D1 and D2 of another p3 from another filamentous phages.

In our design we wanted to keep the signal sequence and D3 of M13, because their are crucial for the formation of the phage. We just want to insert D1 and D2 from another phages (we’ll call it X).

T--Aix-Marseille--P3map.png

Another way to engineered M13, is to remove entierely the protein III from the phage genome and to reconstruct it in another plasmid. Thus, we create another part : p3_D3, which is the domain involved in the assembly and release of M13 particles.

Read more…

Phagemid

As M13KO7 genome is not capable to be used for the phage assemblage, we will use a phagemid which will carry a toxin to assemble our ingeneered M13 phage.

We thought about using the Super Nova toxin (BBa_K1491017), made by the iGEM team Carnegie Mellon in 2014. This toxin has multiple benefits, because of the production of ROS occuring only in a yellow - orange light, we can produce phages-like particules carring this toxin without killing E.coli. However, when this toxin will be produced in X. fastidiosa with the light coming from the sun, the bacterium will be harmed, even if it is in the Xylem vessels. To optimise the production of this toxin in X. fastidiosa we tried to find a strong and constitutive promoter of this bacterium.

We thought about multiple ways to engineer our phagemid.

Read more…