Difference between revisions of "Part:BBa K523013:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
  
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We (the team of EPF Lausanne of 2013) improved this part by characterizing it extensively. In our opinion the images of the eppendorf tubes (see mainpage) aren’t an indisputable proof, that the fusion protein between INP and YFP was in fact on the outer membrane of E.coli. <br>
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We started by using a confocal microscope, the expected outcome was to see fluorescence only at the surface of the bacteria. (image positive control1 compared to image INP-yFp2) But as you can see by comparing Figure 1 and 2, it wasn’t possible to proof surface localization by simply looking at the Bacteria that express the fusion protein. But what we could see was that some bacteria showed clustered YFP. In the literature (reference) we found that the ice nucleation protein forms aggregates in the cell membrane, so we assume we see this phenomena. (image confocal 3 and zoom4)<br>
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We (the team of EPF Lausanne of 2013) improved this part by characterizing it extensively.  
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We determined that approximatively 30% of the cells express the fusion protein.<br>   
+
<br>
 +
In our opinion the images of the eppendorf tubes (see mainpage) was not an indisputable proof, that the fusion protein between INP and YFP was in fact on the outer membrane of E.coli.  
 +
<br>
 +
We started by using a confocal microscope, the expected outcome was to see fluorescence only at the surface of the bacteria. (image positive control1 compared to image INP-yFp2)
 +
<br>
 +
But as you can see by comparing Figure 1 and 2, it wasn’t possible to proof surface localization by simply looking at the Bacteria that express the fusion protein. But what we could see was that some bacteria showed clustered YFP. In the literature (reference) we found that the ice nucleation protein forms aggregates in the cell membrane, so we assume we see this phenomena.  
 +
(image confocal 3 and zoom4)
 +
<br>
 +
We determined that approximatively 30% of the cells express the fusion protein.
 +
<br>   
 
Then we performed an immunofluorescent staining assay using an anti-GFP antibody, which binds also to YFP.  
 
Then we performed an immunofluorescent staining assay using an anti-GFP antibody, which binds also to YFP.  
 
The following results were observed:<br>
 
The following results were observed:<br>
 
(image inp-yfp bf 5, y6, red7, merge8, neg ctl9)<br>
 
(image inp-yfp bf 5, y6, red7, merge8, neg ctl9)<br>
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As you can see in Figures 5-9 the signal from the YFP and from the antibody superimposes nicely. Which proofs that the YFP is on the outer membrane of the cells since otherwise the antibody couldn’t have reached it. <br>
+
As you can see in Figures 5-9 the signal from the YFP and from the antibody superimposes nicely. Which proofs that the YFP is on the outer membrane of the cells since otherwise the antibody couldn’t have reached it.  
 +
<br>
 
With all this proof we concluded that the INP-YFP fusion protein is actually exported to the outer membrane of E.coli.
 
With all this proof we concluded that the INP-YFP fusion protein is actually exported to the outer membrane of E.coli.
  

Revision as of 19:18, 3 October 2013


We (the team of EPF Lausanne of 2013) improved this part by characterizing it extensively.
In our opinion the images of the eppendorf tubes (see mainpage) was not an indisputable proof, that the fusion protein between INP and YFP was in fact on the outer membrane of E.coli.
We started by using a confocal microscope, the expected outcome was to see fluorescence only at the surface of the bacteria. (image positive control1 compared to image INP-yFp2)
But as you can see by comparing Figure 1 and 2, it wasn’t possible to proof surface localization by simply looking at the Bacteria that express the fusion protein. But what we could see was that some bacteria showed clustered YFP. In the literature (reference) we found that the ice nucleation protein forms aggregates in the cell membrane, so we assume we see this phenomena. (image confocal 3 and zoom4)
We determined that approximatively 30% of the cells express the fusion protein.
Then we performed an immunofluorescent staining assay using an anti-GFP antibody, which binds also to YFP. The following results were observed:
(image inp-yfp bf 5, y6, red7, merge8, neg ctl9)
As you can see in Figures 5-9 the signal from the YFP and from the antibody superimposes nicely. Which proofs that the YFP is on the outer membrane of the cells since otherwise the antibody couldn’t have reached it.
With all this proof we concluded that the INP-YFP fusion protein is actually exported to the outer membrane of E.coli.


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