Part:BBa_K2114001

Characterization

This part was used and characterized by the iGEM team Freiburg 2016.

I) Expression analysis by Western blotting

Figure 2: Expression analysis of spore coat proteins.

BBa_K2114001 was assembled into pBS1C [3] alongside with the PCotYZ-RBS promoter (BBa_K2114000) and transformed into competent B. subtilis. Sporulation was induced by starvation in minimal medium. The spore coat proteins were extracted and analysed by SDS-PAGE and Western blotting.

BBa_K2114001 was cloned alongside with The promoter PCotYZ-RBS (BBa_K2114000) into the integration vector pBS1C by 3A assembly. After transformation the cells were selected by chloramphenicol resistance and screened for the disruption of the amyE gene on starch agar plates. Subsequently the positive clones were further cultivated and sporulation was induced by nutrient starvation. The resulting spores were purified from vegetative cells with lysozyme and analyzed by SDS-PAGE and Western blotting. The immunostaining with anti-HA antibodies resulted in the visualization of the expected band at approximately 33 kDa. Additional bands at higher molecular weight were hypothesized to be results from the high cross-linking of spore coat proteins responsible for the enormous rigidity and stability of the spores [4].

II) Verification of surface localizaion by flow cytometry

Figure 3: FACS analysis of the surface-displayed fusion construct. Staining of wild type (not transformed) and engineered (transformed with BBa_K2114001) spores with anti-HA antibodies conjugated to Alexa Fluor 647.

The spores of B. subtilis expressing the part BBa_K2114001 were purified by lysozyme treatment and stained with anti-HA antibodies conjugated to Alexa Fluor® 647 (Cell Signaling Technology®). The antibody could only access surface-localized HA epitopes of the expressed fusion genes and could confirm the successful display of the heterologous protein on the surface of the modified spores while the wild type spores did not exhibit any increase in the fluorescence.

III) Binding of GFP

Figure 4: Evaluation of

To verify the functionality of the expressed fusion construct containing the anti-GFP nanobody the spores were incubated with purified GFP and analyzed by flow cytometry in order to detect the fluorescence. Spore expressing the the part BBa_K2114001 exhibited an additional population with increased fluorescence in comparision to the unmodified wild type spores.

[Representation of the fluorescence in the histogram revealed a clear increase of the fluorescence of modified spores]