Part:BBa_K523013:Experience

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-GFP antibody (ab6658, 1mg/ml) diluted 1:60. The samples were washed 3 times with 200µl PBS. Then the antibody was stained with 2µl avidin daylight labeled (650nm, 1mg/ml) and incubated 1hr on a rotor. Samples were again washed 3 times with 200µl.

Results:
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.

Figure 1 : transformed E.Coli excited at 514 nm(excitation wavelenght for YFP)

Figure 2 : zoom on a single transformed E.Coli excited at 514 nm(excitation wavelenght for YFP)

Another interessant point was cluster of fluorescent dots in the transformed bacteria, that can be seen in image 2. 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 the same was happening when INP was fused to YFP.

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 :

figure 3 :
transformed E.Coli with BBa_K523013 excited to see YFP fluorescence (514nm)

figure 4 :
transformed E.Coli with BBa_K523013 incubated with anti YFP antibody, excited to see antibody antiYFP fluorescence (650nm)

figure 5 :
transformed E.Coli with BBa_K523013;merged image of YFP and antiYFP fluorescence

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-lhttp://intext.nav-links.com/images/dotclear.gifocalize 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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