Team:EPF Lausanne/Project/Materials Methods Drosophila

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=Materials and Methods=
=Materials and Methods=
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We grew E.Coli DH5a transformed with the plasmid C3 containing it (part number, link to parts section). As a negative control we used cultures transformed with the C3 plasmid alone.  
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The Flies.
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A culture volume of 100 ml was spinned down. The pellets as well as the supernatents were used as samples for a protein analysis.  
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The pellets were resuspended in lysis buffer containing urea and sonicated for 15 minutes. For the western blot the samples were run on an SDSpage gel and then transferred to a nitrocellulose membrane. The detection was accomplished using the following antibodies: Anti-his biotin as primary and Streptavidin-HPR as a secondary antibody. Additional to the primary antibody we applied Anti-his HRP.  
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Working with flies: We put the drosophila to sleep with CO2 and then manipulated them with fine paintbrushes and tweezers on a box were we could add CO2 at will.  
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The supernatants were run through a filter device with a 5 kDa cutoff (“centricon”). These samples were also anayzed with a western blot as described above.
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Females / Males: For each experiment we worked exclusively on female flies to have the least variability. We chose to work on females rather than males because the females eat more, as they have to lay eggs, are bigger and have a higher metabolism. We hence assumed that by working with females, we could achieve an higher uptake of bacteria.
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Oregon / Relish: We worked on the drosophila WT Oregon. We also conducted some experiments with the drosophila mutant relish, which is immunodeficient, to see if the basic immune system of the WT flies is harmful to asaia.
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The Infection
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We prepared aliquots of bacteria with OD 100. We then put 50ul of the solution on filters that we placed over the medium which the flies feed on. Therefore the flies take up the bacteria whenever they eat.
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Before infecting the flies we starved them for approximatively two hours in order to ensure that they would  immediately take up the bacteria, and all at approximately the same time.
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The Bacterial Controls
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We infected a set of flies with different types of bacteria whose reaction in the drosophila’s gut is are well known, and therefore use these as controls.
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Positive control (Bacteria that should persist in the gut of the drosophila).
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Pseudomonas entomophila (Pe)
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Erwinia carotovora carotovora 15 (ECC-15)
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Negative control  (Bacteria that do not persist in the gut of drosophila).
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Gac: non persistent Pe mutant
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Time of measurements
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3h: Three hours after the infection we verified that the flies were actually properly infected. We expected to observe the presence of all types of bacteria  (asaia, positive and negative control) as the flies would not have had time to produce an immune response against these bacteria.
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24h: We should start observing a decline in the negative control whilst the positive control persists. We can also start observing the tendency of asaia.
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48h: The positive control should still persist and the negative control should have almost disappeared. We can then compare asaia to the controls and conclude on the persistency of asaia.  
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bove.
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Revision as of 23:14, 24 October 2010



Materials and Methods

The Flies.

Working with flies: We put the drosophila to sleep with CO2 and then manipulated them with fine paintbrushes and tweezers on a box were we could add CO2 at will.

Females / Males: For each experiment we worked exclusively on female flies to have the least variability. We chose to work on females rather than males because the females eat more, as they have to lay eggs, are bigger and have a higher metabolism. We hence assumed that by working with females, we could achieve an higher uptake of bacteria.

Oregon / Relish: We worked on the drosophila WT Oregon. We also conducted some experiments with the drosophila mutant relish, which is immunodeficient, to see if the basic immune system of the WT flies is harmful to asaia.

The Infection

We prepared aliquots of bacteria with OD 100. We then put 50ul of the solution on filters that we placed over the medium which the flies feed on. Therefore the flies take up the bacteria whenever they eat.

Before infecting the flies we starved them for approximatively two hours in order to ensure that they would immediately take up the bacteria, and all at approximately the same time.

The Bacterial Controls

We infected a set of flies with different types of bacteria whose reaction in the drosophila’s gut is are well known, and therefore use these as controls.

Positive control (Bacteria that should persist in the gut of the drosophila). Pseudomonas entomophila (Pe) Erwinia carotovora carotovora 15 (ECC-15)

Negative control (Bacteria that do not persist in the gut of drosophila). Gac: non persistent Pe mutant

Time of measurements

3h: Three hours after the infection we verified that the flies were actually properly infected. We expected to observe the presence of all types of bacteria (asaia, positive and negative control) as the flies would not have had time to produce an immune response against these bacteria.

24h: We should start observing a decline in the negative control whilst the positive control persists. We can also start observing the tendency of asaia.

48h: The positive control should still persist and the negative control should have almost disappeared. We can then compare asaia to the controls and conclude on the persistency of asaia. bove.

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