Difference between revisions of "Team:Aix-Marseille/M13 test"

(→‎Test with Arabidopsis thaliana)
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[[File:T--Aix-Marseille--Leaf-1.png|800px|center|thumb|Fluorescent PLPs in ''Arabidopsis thaliana'' leaf. A) Control leaf without fluorescent PLPs, B) Leaf pricked with fluorescent PLPs, C) Leaf infiltrated with fluorescent PLPs, D) Leaf spreayed with fluorescent PLPs and E) Leaf soaked with fluorescent PLPs.]]
 
[[File:T--Aix-Marseille--Leaf-1.png|800px|center|thumb|Fluorescent PLPs in ''Arabidopsis thaliana'' leaf. A) Control leaf without fluorescent PLPs, B) Leaf pricked with fluorescent PLPs, C) Leaf infiltrated with fluorescent PLPs, D) Leaf spreayed with fluorescent PLPs and E) Leaf soaked with fluorescent PLPs.]]
  
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[[File:T--Aix-Marseille--Leaf-MO-2.png|400px|right|thumb|Fluorescent PLPs seen in ''Arabidopsis thaliana'' leaf petiole by confocal microscopy. Left shows fluorescent PLPs in epithelium and right shows them in plant vessels.]]
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[[File:T--Aix-Marseille--Leaf-MO-2.png|400px|right|thumb|Fluorescent PLPs seen in ''Arabidopsis thaliana'' leafs by confocal microscopy. Left shows fluorescent PLPs in epithelial cells and right shows them in plant vessels.]]
  
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The [[Team:Aix-Marseille/HP/Surveys|farmer's surveys]] advises us that depending on the agriculture form, various final forms of our product were preferable. So we thought that it was preferable to find the best way to administer our cure. In order to find the best way to give the cure to the tree we tested multiple ways. First, we link our phage-like particles (PLPs) to a fluorochrome, thus making a solution of fluorescent PLPs. Then, this solution was used on ''Arabidopsis thaliana'' in three ways :
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The [[Team:Aix-Marseille/HP/Surveys|farmer's surveys]] indicated that for user acceptability, the application method for our treatment was important.  
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* the use of a sting on the leaf (which mimics the injection).
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In order to find possible ways of applying the treatment to a tree we tested multiple ways with our model plant ''Arabidopsis thaliana''.  
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* infiltration of the solution through the leaf.
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To observe the movement of PLP is plants, we attached a fluoreochrome to our phage-like particles (PLPs), making a solution of fluorescent PLPs. Then, this solution was used on ''Arabidopsis thaliana'' in different ways :
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* dropping the solution on the leaf (mimic spray).
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* pricking the leaf (which mimics the injection).
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* the soaking of the solution by the petiol.
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* infiltrating the solution into the leaf.
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The control is leaf without any solution. As you can see the use of sting and infiltration are more efficient than dropping the solution.
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* putting a drop of solution on the leaf (to mimic a spray).
 +
* soaking the petiole of a leaf in the solution.
 +
The control is leaf without any fluorescent PLP.  
 +
As you can see the use of pricking, infiltration and soaking the petiole are all more efficient than using a drop.
  
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Finally, we wanted to know if the phage was able to infiltrate the integrity of the leaf. We can see that all the cells are infiltrated with fluorescent PLPs. Thus, PLPs aren't just confined to leaf vessels.
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Finally, we wanted to know if the phage was able to circulate to different parts of the leaf.  
 +
We can see that all the cells become fluorescent.  
 +
Thus, PLPs are not confined to leaf vessels but can circulate freely in the plant.
  
 
==PLPs rate of production==
 
==PLPs rate of production==
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[[File:T--Aix-Marseille--Recombination.png|400px|right|]]
 
[[File:T--Aix-Marseille--Recombination.png|400px|right|]]
  
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Thanks to [[Team:Aix-Marseille/HP/Interviews|Jacques VAN HELDEN interview]] we put attention to the production rate of our PLPs and the risk of recombination between M13KO7 and the phagemid. This question matter because we aim to create PLPs that aren't able to replicate and thus spread in the environment. Therefore, we test this possibility along our [[Team:Aix-Marseille/Modelmodelling part|model]].
+
Thanks to the interview with [[Team:Aix-Marseille/HP/Interviews|Jacques VAN HELDEN]] we considered the production rate of our PLPs and the risk of recombination between M13KO7 and the phagemid.  
 +
This question matters because we aim to create PLPs that are unable to replicate and thus spread in the environment.  
 +
Therefore, we tested this possibility in the lab and also with our[[Team:Aix-Marseille/Model|model]].
  
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As you can witness, recombination only occurs for 0,1% of phages, this is coherent with our assumptions. However, PLPs represent only 14,2% of the production against 85,7% for the replicative phage. This means that the vast majority of our production is phages containing M13KO7 DNA and that they cannot inject toxins.
+
As you can see, recombination is very rare and only occurs for 0,1% of phages, this is coherent with our assumptions.  
 +
However unfortunately in these tests, PLPs represent only 14,2% of the production against 85,7% for the replicative phage.  
 +
This means that the vast majority of our production is phages containing M13KO7 DNA and that they cannot inject toxins.
  
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Eventually, this result is due because the M13KO7 M13ori has a stretch with a better for p5. To counteract this result, we could make p5 select the phagemid by modifying more M13KO7 M13ori, or we could try to enhance the M13ori part of our phagemid.
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<!-- THIS DOES NOT MAKE SENSE JAMES
 +
This result is due because the M13KO7 M13ori has a stretch with a better for p5. To counteract this result, we could make p5 select the phagemid by modifying more M13KO7 M13ori, or we could try to enhance the M13ori part of our phagemid.
 +
-->

Revision as of 15:06, 1 November 2017

Phage-like particles tests

Phage-like particles (PLPs) in the soil

T--Aix-Marseille--Pinsoil-2.png

We first tested the lifetime of our phage-like particles (PLPs) in the soil. The soil samples can from the [http://www.moulindevelaux.com/ Moulin de Velaux] which is an olive grower cooperative in southern France. For this experiment, we put M13 phages in the soil and we let it incubate for different amounts of time. As seen in figure 1, the population of phage decreases with time because they are degraded by micro-organisms that lived in the soil. The experimental half-life of our phage-like particles is approximately of 4 hours.

With this result, we can be confident that our phage-like particles solution doesn't pollute the soil. This test is one of the many tests needed for the marketing authorization procedure of our production. Check out our legislation page for more information.

Test with Arabidopsis thaliana

Fluorescent PLPs in Arabidopsis thaliana leaf. A) Control leaf without fluorescent PLPs, B) Leaf pricked with fluorescent PLPs, C) Leaf infiltrated with fluorescent PLPs, D) Leaf spreayed with fluorescent PLPs and E) Leaf soaked with fluorescent PLPs.
Fluorescent PLPs seen in Arabidopsis thaliana leafs by confocal microscopy. Left shows fluorescent PLPs in epithelial cells and right shows them in plant vessels.

The farmer's surveys indicated that for user acceptability, the application method for our treatment was important. In order to find possible ways of applying the treatment to a tree we tested multiple ways with our model plant Arabidopsis thaliana. To observe the movement of PLP is plants, we attached a fluoreochrome to our phage-like particles (PLPs), making a solution of fluorescent PLPs. Then, this solution was used on Arabidopsis thaliana in different ways :

  • pricking the leaf (which mimics the injection).
  • infiltrating the solution into the leaf.
  • putting a drop of solution on the leaf (to mimic a spray).
  • soaking the petiole of a leaf in the solution.

The control is leaf without any fluorescent PLP. As you can see the use of pricking, infiltration and soaking the petiole are all more efficient than using a drop.

Finally, we wanted to know if the phage was able to circulate to different parts of the leaf. We can see that all the cells become fluorescent. Thus, PLPs are not confined to leaf vessels but can circulate freely in the plant.

PLPs rate of production

T--Aix-Marseille--Recombination.png

Thanks to the interview with Jacques VAN HELDEN we considered the production rate of our PLPs and the risk of recombination between M13KO7 and the phagemid. This question matters because we aim to create PLPs that are unable to replicate and thus spread in the environment. Therefore, we tested this possibility in the lab and also with ourmodel.

As you can see, recombination is very rare and only occurs for 0,1% of phages, this is coherent with our assumptions. However unfortunately in these tests, PLPs represent only 14,2% of the production against 85,7% for the replicative phage. This means that the vast majority of our production is phages containing M13KO7 DNA and that they cannot inject toxins.