Team:EPF Lausanne/Project droso

From 2010.igem.org

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(Conclusion to the Drosophila experiments)
 
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[[Image:Mousquito and asaia.png|right|200px|caption]]
[[Image:Mousquito and asaia.png|right|200px|caption]]
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The final goal of our project is for our modified Asaia to survive and produce proteins in the mosquito's gut. However, working with mosquitoes requires special equipment that we do not have at EPFL. We wondered if we could work on another insect which is less demanding, and therefore turned towards Drosophila (commonly known as the fruit fly), which is much easier to work with.
+
The final goal of our project is for our modified Asaia to survive and produce proteins in the mosquito's gut. However, working with mosquitoes requires special equipment that we do not have at EPFL. We wondered if we could work on another insect which is less demanding, and therefore turned towards ''Drosophila'' (commonly known as the fruit fly), which is much easier to work with.
-
Considering the fact that bacteria that live in the guts of insects are not very common, we assumed that there was a fair chance that Asaia could persist in Drosophila and that we could use the it as an alternative to mosquitoes for our basic experiments.
+
Considering the fact that bacteria that live in the guts of insects are not very common, we assumed that there was a fair chance that Asaia could persist in ''Drosophila'' and that we could use it as an alternative to mosquitoes for our basic experiments.
-
We therefore decided to work in a first phase with Drosophila, which we discuss in the first part of this section, and finally turn to mosquitoes, which we discuss in the second part of this section.
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We therefore decided to initially work with ''Drosophila'', which we discuss in the first part of this section, and later turn to mosquitoes, which we discuss in the second part of this section.
= I) Experiments on ''Drosophila'' =
= I) Experiments on ''Drosophila'' =
   
   
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Using ''Drosophila melanogaster''  we aimed to address two questions:
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With ''Drosophila melanogaster''  we aimed to address two questions:
<br> i. Is ''Asaia'' toxic for ''Drosophila''?  
<br> i. Is ''Asaia'' toxic for ''Drosophila''?  
<br> ii.Is ''Asaia'' able to colonize the Drosophila'' gut and persist?  
<br> ii.Is ''Asaia'' able to colonize the Drosophila'' gut and persist?  
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===1) ''Asaia'' is not toxic for Drosophila===
===1) ''Asaia'' is not toxic for Drosophila===
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[[Image:Survival.png|center|600px|thumb|bottom|'''Figure 2''' ''Asaia'' is not toxic to ''Drosophia''. Immunodeficient ''Relish'' (A) and wild-type ''Oregon'' (B) flies were infected with different bacterial strains. A lethal control strain (''P. entomophila''), a non-lethal control strain (''Ecc 15'') and our ''Asaia'' bacteria. For these experiments we used two different fly strains (''Oregon'' and ''Relish''). Deaths were counted over the course of the experiment..]]
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[[Image:Survival.png|center|600px|thumb|bottom|'''Figure 2''' ''Asaia'' is not toxic to ''Drosophila''. Immunodeficient ''Relish'' (A) and wild-type ''Oregon'' (B) flies were infected with different bacterial strains. A lethal control strain (''P. entomophila''), a non-lethal control strain (''Ecc 15'') and our ''Asaia'' bacteria. Dead flies were counted over the course of the experiment..]]
We infected ''Drosophila'' with different [https://2010.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Project/Materials_Methods_Drosophila bacterial strains], a pathogenic control starin (''P. entomophila''), a non-pathogenic control strain (Ecc 15) and our ''Asaia'' bacteria. For these experiments we used two different [https://2010.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Project/Materials_Methods_Drosophila fly strains] (''Oregon'' and ''Relish'').  
We infected ''Drosophila'' with different [https://2010.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Project/Materials_Methods_Drosophila bacterial strains], a pathogenic control starin (''P. entomophila''), a non-pathogenic control strain (Ecc 15) and our ''Asaia'' bacteria. For these experiments we used two different [https://2010.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Project/Materials_Methods_Drosophila fly strains] (''Oregon'' and ''Relish'').  
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We found that ''Asai''a does not cause significantly more deaths than the non-pathogenic bacteria or in the uninfected control (Figure 2).
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We found that ''Asaia'' does not cause significantly more deaths than the non-pathogenic bacteria or the uninfected control (Figure 2).
===2) ''Asaia'' is not persistent in ''Drosophila'' ===
===2) ''Asaia'' is not persistent in ''Drosophila'' ===
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[[Image:Persistance.png|center|400px|thumb|bottom|'''Figure 3''' ''Asaia'' does not persist in ''Drosophila''.]]
[[Image:Persistance.png|center|400px|thumb|bottom|'''Figure 3''' ''Asaia'' does not persist in ''Drosophila''.]]
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With this last experiment we wanted to ascertain wether ''Asaia'' persisted in ''Drosophila'' and monitore how many ''Asaia'' were present in the ''Drosophila’s'' gut after 3h, 24h and 48h.  
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With this last experiment we wanted to ascertain wether ''Asaia'' persisted in ''Drosophila'' and monitor how many ''Asaia'' were present in the ''Drosophila’s'' gut after 3h, 24h and 48h.  
To be able to “count” the number of ''Asaia'' at these three different periods of time, we had to retrieve the bacteria inside the flies, plate them and after incubation count the number of colonies present.  
To be able to “count” the number of ''Asaia'' at these three different periods of time, we had to retrieve the bacteria inside the flies, plate them and after incubation count the number of colonies present.  
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The survival assay reveals that "Asaia" is not lethal for "Drosophila".  
The survival assay reveals that "Asaia" is not lethal for "Drosophila".  
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The persistence assay showed that "Asaia" is unable to establish itself in the fruit fly's gut. This prevents us to use "Drosophila" as a substitute to mosquitoes.
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The persistence assay showed that "Asaia" is unable to establish itself in the fruit fly's gut. This prevents us to use "Drosophila" as a model for Asaia insect interactions.  
As mentioned before, bacteria that live in insects’ gut are not common and it is possible that because it cannot survive in "Drosophila", it is actually very specific to mosquitoes. With this assumption, we could assume that we can introduce our modified "Asaia" into the wildlife with very little risk of it spreading to other insects.
As mentioned before, bacteria that live in insects’ gut are not common and it is possible that because it cannot survive in "Drosophila", it is actually very specific to mosquitoes. With this assumption, we could assume that we can introduce our modified "Asaia" into the wildlife with very little risk of it spreading to other insects.
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= II) Experiments on mosquitoes =
= II) Experiments on mosquitoes =
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The final objective of this project is to determine if mosquitoes carrying our engineered Asaia are less capable of transmitting malaria. However, we do not have mosquito swarms here in EPFL. To do this experiment, we contacted the Institute Pasteur in Paris to collaborate with us and proceed with our experiments. Nevertheless, the experiments will not be done before the iGEM jamboree, so we cannot show results. We are still discussing with them on the experiments and on how we are going to proceed.
+
The final objective of this project is to determine if mosquitoes carrying our engineered Asaia are less capable of transmitting malaria. However, we do not have mosquito swarms here in EPFL. To do this experiment, we contacted the Institute Pasteur in Paris to collaborate with us and proceed with our experiments. Nevertheless, the experiments will not be done before the iGEM jamboree...
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Latest revision as of 19:09, 27 October 2010



Contents

Introduction

caption

The final goal of our project is for our modified Asaia to survive and produce proteins in the mosquito's gut. However, working with mosquitoes requires special equipment that we do not have at EPFL. We wondered if we could work on another insect which is less demanding, and therefore turned towards Drosophila (commonly known as the fruit fly), which is much easier to work with.

Considering the fact that bacteria that live in the guts of insects are not very common, we assumed that there was a fair chance that Asaia could persist in Drosophila and that we could use it as an alternative to mosquitoes for our basic experiments.

We therefore decided to initially work with Drosophila, which we discuss in the first part of this section, and later turn to mosquitoes, which we discuss in the second part of this section.

I) Experiments on Drosophila

With Drosophila melanogaster we aimed to address two questions:
i. Is Asaia toxic for Drosophila?
ii.Is Asaia able to colonize the Drosophila gut and persist?

(See Materials and Methods for details on how the experiments were conducted.)

Our main results

Figure 1 Observing Asaia that express GFP in vivo


1) Asaia is not toxic for Drosophila

Figure 2 Asaia is not toxic to Drosophila. Immunodeficient Relish (A) and wild-type Oregon (B) flies were infected with different bacterial strains. A lethal control strain (P. entomophila), a non-lethal control strain (Ecc 15) and our Asaia bacteria. Dead flies were counted over the course of the experiment..

We infected Drosophila with different bacterial strains, a pathogenic control starin (P. entomophila), a non-pathogenic control strain (Ecc 15) and our Asaia bacteria. For these experiments we used two different fly strains (Oregon and Relish).

We found that Asaia does not cause significantly more deaths than the non-pathogenic bacteria or the uninfected control (Figure 2).

2) Asaia is not persistent in Drosophila

Figure 3 Asaia does not persist in Drosophila.

With this last experiment we wanted to ascertain wether Asaia persisted in Drosophila and monitor how many Asaia were present in the Drosophila’s gut after 3h, 24h and 48h.

To be able to “count” the number of Asaia at these three different periods of time, we had to retrieve the bacteria inside the flies, plate them and after incubation count the number of colonies present.

To do this the flies were crushed in the medium corresponding to the bacteria we were interested in (i.e: Gly for Asaia, LB for Pe, etc). We then did a serial dilution eleven times with a factor of ten with the crushed flies, and plated each dilution with antibiotics to specifically select the bacteria we were interested in.

Conclusion

The survival assay reveals that "Asaia" is not lethal for "Drosophila".

The persistence assay showed that "Asaia" is unable to establish itself in the fruit fly's gut. This prevents us to use "Drosophila" as a model for Asaia insect interactions.

As mentioned before, bacteria that live in insects’ gut are not common and it is possible that because it cannot survive in "Drosophila", it is actually very specific to mosquitoes. With this assumption, we could assume that we can introduce our modified "Asaia" into the wildlife with very little risk of it spreading to other insects.

II) Experiments on mosquitoes

The final objective of this project is to determine if mosquitoes carrying our engineered Asaia are less capable of transmitting malaria. However, we do not have mosquito swarms here in EPFL. To do this experiment, we contacted the Institute Pasteur in Paris to collaborate with us and proceed with our experiments. Nevertheless, the experiments will not be done before the iGEM jamboree...


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