Part:BBa_K1457000:Experience

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Applications of BBa_K1457000

iGEM Marburg 2015 further improved this part.

Westernblot

Figure 1: Westernblot shows the presence of a His taged fusion protein consisting of Spycatcher-GFP

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Figure 2: Westernblot shows the binding of the GFP-spycatcher (BBa_K1650048) to CsgA-spytag (BBa_K1650047). Both constructs were cotransformed in the same cells. The band at 55kd is signifantly higher than the band of spycatcher-GFP-His alone at 35-40kd proving that spycatcher and spytag have bound intracellulary

Congo red and chrystal violet assays

Figure 1: Congo red is a dye that stains amyloid fibres such as curlis. The negativ control shows that cells without BBa_K1650047 are not stained thus are not expressing CsgA

Figure 2: Additional to congo red, chrystal violet is used to stain biofilms based on curlis. Measurements are done using a plate reader.

Westernblot

Figure 3: Westernblot shows the binding of the GFP-spycatcher (BBa_K1650048) to CsgA-spytag (BBa_K1650047). Both constructs were cotransformed in the same cells. The band at 55kd is signifantly higher than the band of spycatcher-GFP-His alone at 35-40kd proving that spycatcher and spytag have bound intracellulary

Fluorescence Microscopie

Figure 4: E.coli cells carrying BBa_K1650047, thus expressing CsgA-spytag, are grown in 24-well-plates to form a biofilm. Biofilm is washed multiple times with PBS to remove medium.
Celllysate of cells carrying BBa_K1650046 is prepared to obtain GFP-spycatcher. Celllysate containing GFP-spycatcher is poured onto the biofilm. To remove unspecific bindings, cells are washed again with PBS.
Pictures show from left to right: Overlay of phasecontrast picture and green fluorescence, phasecontrast picture alone and green fluorescence alone. Only green fluorescent cells express CsgA-spytag which is bound by GFP-spycatcher

Scanning Electron Microscopie

Figure 5: Scanning Electron Microscopie shows Curli fibers displayed on the cell surface

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