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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==<font size = "5">Futher Characteristation by iGEM13_EPF_Lausanne</font>==
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As we (EPFL 2013 team) used the Biobrick BBa_K523013 for our project, we 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 experiments with a confocal and also a inverted (NIKON eclipse it) microscope.
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<font size = "4">Process:</font>
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<br>Competent cells were transformed with BBa_K523013 and grown on a plate with chloramphenicol antibiotic. From these transformed cells we made a glycerol stock and worked with plated cells from this glycerol stock.
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<br>For each experiment, cells were inoculate overnight at 37°C, in 3ml LB +chloramphenicol medium. Then cells were diluted to 0.3 OD and grown until 0.7-0.8 OD. Then they were pelleted and resuspended in 200µl PBS.
 
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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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<br> '''1)''' To visualize the protein fluorescence, we directly used this solution.
 
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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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<br>'''2)''' To confirm the localization of the protein at the cell surface, this solution of cells was incubated 1hr in a rotor, with 10µl of an anti-YFP antibody (ab6658, 1mg/ml) diluted 1:60. The samples were washed 3 times with 200µl PBS. Then the antibody was stained with 2µl avidin dylight labeled (650nm, 1mg/ml) and incubated 1hr on a rotor. Samples were again washed 3 times with 200µl.
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<font size = "4">Results:</font>
 
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Images : (negative control and samples)
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<br>The protein fluorescence was directly observed with a confocal microscope (Figure1) and an inverted microscope. We determined approximately 30% of the cells to be fluorescent. In the transformed cells, cluster of fluorescent specks can be seen (Figure 2). This is consistant with fact that we found in the literature ([http://www.ncbi.nlm.nih.gov/pubmed/?term=Clustering+of+ice+nucleation+protein+correlates [1]]) that the ice nucleation protein forms aggregates in the cell membrane. With the confocal microscope we expected to distinguish if the fluorescence was at the cell surface, but we were not able to make any difference between the cell surface and cytoplasmic expression.
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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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[[File:EPF-lausanne_fluoYFP.jpg|thumb|290px|left|Figure 1 : Confocal image of BBa_K523013 transformed DH5alpha E.coli, excited at 514 nm(excitation wavelenght for YFP)]]
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[[File:EPF-lausanne_fluoYFPdots.jpg|thumb|310px|center|Figure 2 : Confocal image of a zoom on a single BBa_K523013 transformed DH5alpha E.Coli, excited at 514 nm(excitation wavelenght for YFP)]]
  
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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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So, to have a clear idea of the localization, we treated cells as explained in "Process 2)" and went to look at them under the inverted microscope.
 
The following results were observed :  
 
The following results were observed :  
 
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[[File:EPF-lausanne_brightfield.jpg|thumb|250px|left|figure 1 : <br> transformed E.Coli with BBa_K523013 excited to see YFP fluorescence (514nm)]]
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[[File:EPF-lausanne_brightfield.jpg|thumb|270px|left|Figure 3 : <br> Inverted microscope composite image of BBa_K523013 transformed DH5alpha E.coli showing YFP fluorescence (514nm)]]
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[[File:EPF-lausanne_ab.jpg|thumb|250px|center|figure 2 : <br> transformed E.Coli with BBa_K523013 incubated with anti YFP antibody, excited to see antibody antiYFP fluorescence ]]
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[[File:EPF-lausanne_ab.jpg|thumb|260px|left|Figure 4 : <br> Inverted microscope composite  image of BBa_K523013 transformed DH5alpha E.coli,  stained with biotinylated anti-YFP antibody and avidin dylight (650nm) ]]
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[[File:EPF-lausanne_merged.jpg|thumb|250px|right|figure 3 : <br> transformed E.Coli with BBa_K523013;merged image of YFP and antiYFP fluorescence]]
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[[File:EPF-lausanne_merged.jpg|thumb|230px|left|Figure 5 : <br> <font size = "0.5">Inverted microscope composite  image of BBa_K523013 transformed DH5alpha E.coli,  stained with biotinylated anti-YFP antibody and avidin dylight (650nm). The composite image is a merge of the YFP fluorescence image and the antibody fluorescence image</font> ]]
  
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As we can see, the antibody binds 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 the protein was inside the cell. http://intext.nav-links.com/images/dotclear.gif
 
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With all these proofs we concluded that the INP-YFP fusion protein is actually exported to the outer membrane of E.coli.
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<br> <br>
  
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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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===Applications of BBa_K523013===
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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.
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BBa_K523013 is function to display YFP at bacterial cell surface.
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===Applications of BBa_K523013===
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<br>The cloning strategy can thus be used to display any protein as big as 60kDa to the Ice Nucleation Protein [http://www.ncbi.nlm.nih.gov/pubmed/12480350].
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 23:28, 4 October 2013




Futher Characteristation by iGEM13_EPF_Lausanne

As we (EPFL 2013 team) used the Biobrick BBa_K523013 for our project, we 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 experiments with a confocal and also a inverted (NIKON eclipse it) microscope.

Process:
Competent cells were transformed with BBa_K523013 and grown on a plate with chloramphenicol antibiotic. From these transformed cells we made a glycerol stock and worked with plated cells from this glycerol stock.
For each experiment, cells were inoculate overnight at 37°C, in 3ml LB +chloramphenicol medium. Then cells were diluted to 0.3 OD and grown until 0.7-0.8 OD. Then they were pelleted and resuspended in 200µl PBS.

1) To visualize the protein fluorescence, we directly used this solution.

2) To confirm the localization of the protein at the cell surface, this solution of cells was incubated 1hr in a rotor, with 10µl of an anti-YFP antibody (ab6658, 1mg/ml) diluted 1:60. The samples were washed 3 times with 200µl PBS. Then the antibody was stained with 2µl avidin dylight labeled (650nm, 1mg/ml) and incubated 1hr on a rotor. Samples were again washed 3 times with 200µl.



Results:

The protein fluorescence was directly observed with a confocal microscope (Figure1) and an inverted microscope. We determined approximately 30% of the cells to be fluorescent. In the transformed cells, cluster of fluorescent specks can be seen (Figure 2). This is consistant with fact that we found in the literature ([http://www.ncbi.nlm.nih.gov/pubmed/?term=Clustering+of+ice+nucleation+protein+correlates [1]]) that the ice nucleation protein forms aggregates in the cell membrane. With the confocal microscope we expected to distinguish if the fluorescence was at the cell surface, but we were not able to make any difference between the cell surface and cytoplasmic expression.

Figure 1 : Confocal image of BBa_K523013 transformed DH5alpha E.coli, excited at 514 nm(excitation wavelenght for YFP)
Figure 2 : Confocal image of a zoom on a single BBa_K523013 transformed DH5alpha E.Coli, excited at 514 nm(excitation wavelenght for YFP)


So, to have a clear idea of the localization, we treated cells as explained in "Process 2)" and went to look at them under the inverted microscope. The following results were observed :

Figure 3 :
Inverted microscope composite image of BBa_K523013 transformed DH5alpha E.coli showing YFP fluorescence (514nm)
Figure 4 :
Inverted microscope composite image of BBa_K523013 transformed DH5alpha E.coli, stained with biotinylated anti-YFP antibody and avidin dylight (650nm)
Figure 5 :
Inverted microscope composite image of BBa_K523013 transformed DH5alpha E.coli, stained with biotinylated anti-YFP antibody and avidin dylight (650nm). The composite image is a merge of the YFP fluorescence image and the antibody fluorescence image





















As we can see, the antibody binds 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 the protein was inside the cell. http://intext.nav-links.com/images/dotclear.gif
With all these proofs we concluded that the INP-YFP fusion protein is actually exported to the outer membrane of E.coli.

Applications of BBa_K523013

BBa_K523013 is function to display YFP at bacterial cell surface.
The cloning strategy can thus be used to display any protein as big as 60kDa to the Ice Nucleation Protein [http://www.ncbi.nlm.nih.gov/pubmed/12480350].

User Reviews

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