Difference between revisions of "Part:BBa K523013:Experience"
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As we (EPFL 2013 team) used the Biobrick BBa_K523013 for our project, we have decided to improve it by better characterizing it. The group that made it was not able to prove with certainty that the YFP was exported to the outer membrane of E.Coli thanks to INP. In order to see the location of this fluorescent protein, we performed microscopy with a confocal microscope.  
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We (the team of EPF Lausanne of 2013) improved this part by characterizing it extensively.  
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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.  
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Competent cells were transformed with this part and grown on a plate with the correct antibiotic. After overnight growth, colonies were picked and dissolved in LB, then grown until an OD of between 0.6 and 0.8.  
 
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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)
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The fluorescence was directly observed with a YFP filter. We expected to see fluorence only at the membrane, but the resolution was not sufficient to see where exactly the fluorence was located. We determind approximately 30% of the cells to be fluorescent.The negative control was not fluorescent, which was a good point.
 
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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.
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Images : (negative control and samples)
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(image confocal 3 and zoom4)
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We determined that approximatively 30% of the cells express the fusion protein.
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Another interessant point was cluster of fluorescent dots in the transformed bacteria. In the literature ([http://www.ncbi.nlm.nih.gov/pubmed/?term=Clustering+of+ice+nucleation+protein+correlates [1]]) 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)
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Then we performed an immunofluorescent staining assay using an anti-GFP antibody, which binds also to YFP.  
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The following results were observed:<br>
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(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.  
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Then, to have a clear idea of the localization, cells were incubated with an anti-GFP antibody (YFP and GFP being really close, antibodies can be used for both molecules) and washed several times after.  
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The following results were observed :  
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images : (negative control and sample)
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As we can see, the antibody binds at the outside of the bacteria, since it is way to large to enter the cells. The points nicely co-localize with the YFP fluorescence and prove its external localization, since otherwise antibody could not have reached it if it was inside the cell.
 
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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 15:01, 4 October 2013


As we (EPFL 2013 team) used the Biobrick BBa_K523013 for our project, we have decided to improve it by better characterizing it. The group that made it was not able to prove with certainty that the YFP was exported to the outer membrane of E.Coli thanks to INP. In order to see the location of this fluorescent protein, we performed microscopy with a confocal microscope.
Competent cells were transformed with this part and grown on a plate with the correct antibiotic. After overnight growth, colonies were picked and dissolved in LB, then grown until an OD of between 0.6 and 0.8.
The fluorescence was directly observed with a YFP filter. We expected to see fluorence only at the membrane, but the resolution was not sufficient to see where exactly the fluorence was located. We determind approximately 30% of the cells to be fluorescent.The negative control was not fluorescent, which was a good point.
Images : (negative control and samples)
Another interessant point was cluster of fluorescent dots in the transformed bacteria. In the literature ([http://www.ncbi.nlm.nih.gov/pubmed/?term=Clustering+of+ice+nucleation+protein+correlates [1]]) 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)


Then, to have a clear idea of the localization, cells were incubated with an anti-GFP antibody (YFP and GFP being really close, antibodies can be used for both molecules) and washed several times after. The following results were observed :
images : (negative control and sample)

As we can see, the antibody binds at the outside of the bacteria, since it is way to large to enter the cells. The points nicely co-localize with the YFP fluorescence and prove its external localization, since otherwise antibody could not have reached it if it was inside the cell.
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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