Part:BBa_K1457000:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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

7

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

User Reviews

UNIQ8f7b73b2b18d996a-partinfo-00000001-QINU UNIQ8f7b73b2b18d996a-partinfo-00000002-QINU