Team:EPF Lausanne/Project droso

From 2010.igem.org

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[[Image:Mousquito and asaia.png|center|200px|caption]]
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=Introduction=
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= Experiments on Drosophilia =
 
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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. Working with mosquitoes however requires special equipment that we do not have at EPFL, and we wondered if we could work on another insect which is less demanding. We therefore turned towards Drosophila, commonly known as the fruit fly, which is much easier to work with.
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[[Image:Mousquito and asaia.png|right|200px|caption]]
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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 mosquitos for our basic experiments.
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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.
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Using Drosophila melanogaster  we aimed to address two questions:
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i) Is Asaia pathogenic for Drosophila?
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ii) Is Asaia able to colonize the Drosophila gut and persist?
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= Our main results =
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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.
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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.
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[[mage:Asaiaflies.png|center|100px|''Figure 1'''Observing Asaia that express GFP in vivo]]
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= I) Experiments on ''Drosophila'' =
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With ''Drosophila melanogaster''  we aimed to address two questions:
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===Toxicity of Asaia===
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<br> i. Is ''Asaia'' toxic for ''Drosophila''?
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<br> ii.Is ''Asaia'' able to colonize the Drosophila'' gut and persist?
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[[Image:Survival.png|600px|thumb|bottom|'''Figure 2''' Asaia is not toxic to Drosophila. Relish (A) and Oregon (B) flies were infected with different bacterial strains and monitored them over time. Deaths were counted and added up over the course of the experiment.]]
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(See [https://2010.igem.org/wiki/index.php?title=Team:EPF_Lausanne/Project/Materials_Methods_Drosophila Materials and Methods] for details on how the experiments were conducted.)
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To find out if Asaia is toxic for Drosophila and if it can persist, we fed Asaia to flies. If we fed Asaia that expressed GFP to our flies, we could observe the fluorescent bacteria in the gut of the flies (Figure 1).
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== Our main results ==
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Then we moved on to find out more quantitatively if Asaia was toxic for our flies. 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). The strain Relish does not have an immune system. We found that Asaia does not cause significantly more deaths than the non-pathogenic bacteria or in the uninfected control (Figure 2).
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[[Image:Asaiaflies.png|center|600px|thumb|bottom|'''Figure 1''' Observing ''Asaia'' that express GFP in vivo]]
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==Persistancy essay==
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===1) ''Asaia'' is not toxic for Drosophila===
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===Observing the bacteria in vivo===
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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..]]
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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 infected flies with asaia expressing GFP and observed them under a microscope with a GFP filter over a period of three days. We hoped this would enable us to determine wether the asaia were persistent in the flies.
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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).
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Controls: In addition to the GFP filter we observed the flies with a DSR filter to discard natural fluorescence. We worked with both oregon and relish flies, and we infected an extra set with Pe and ECC (positive controls). Negative control: flies not infected with GFP expressing bacteria?
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===2) ''Asaia'' is not persistent in ''Drosophila'' ===
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Conclusion: It is hard to make a good conclusion with the results of these experiments, primarily because the flies’ eggs are naturally fluorescent and it was not always obvious to determine the origin of the fluorescence.
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[[Image:Persistance.png|center|400px|thumb|bottom|'''Figure 3''' ''Asaia'' does not persist in ''Drosophila''.]]
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The results were negative for a the experience and we repeteated it 4 times.
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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.  
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''graphe des drosophiles''
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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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===Plating the bacteria and quantizing the bacteria present in the flies’ gut===
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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.
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[[Image:Persistance.png|600px|thumb|bottom|'''Figure 3''' Asaia does not  persist in Drosophila.]]
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==Conclusion==
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With this last experiment we wanted to ascertain wether asaia persisted in drosophila and quantize how many asaia were present in the drosophila’s gut after 3h, 24h and 48h.  
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The survival assay reveals that "Asaia" is not lethal for "Drosophila".  
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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.  
+
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.  
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To do this we disinfected the exterior of the flies by washing them with ethanol (for less than five seconds) and then rinsing them with water. The flies were then crushed in the medium corresponding to the bacteria we were interested in (i.e: Gly+LB 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.
+
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 =
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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...
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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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