Designed by: Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Buescher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schaefer, Carolin Schmelas, Silvan Schmitz, Max Waldhauer Group: iGEM14_Heidelberg (2014-10-10)
This part is the N-terminal splicing half in a split sfGFP intein splicing assay. The C-terminal splicing partner is BBa_K1362173, the N- and C-terminal non-splicing control halves are BBa_K1362174 and BBa_K1362172, respectively. BBa_K1362170 served as positive control.
The assay was used to both characterize the reconstitution of split superfold GFP (BBa_I746908) as well as to demonstrate the splicing activity of the NpuDnaE split Intein BBa_K1362400.
Visit the Heidelberg 2014's wiki for a detailed documentation or find a summary of our results in the experience section of this part.
Split sfGFP reconstitution
Figure 1: Cloning Strategy for the bicistronic expression of the two parts of split sfGFP. GFP_N was amplified from BBa_K1362173, CPEC overhangs and the NpuDnaE_N were in the PCR primer. NpuDnaE_C was amplified from BBa_K1362171 with Oligos containing CPEC overhangs and the GFP_C. Both PCR products were cloned in a biscistronic expression backbone using CPEC.
Figure 2: Successful in vivo restorarion of sfGFP fluorescence. Fluorecence intensities detected at 475nm exitation and 512 nm emission wavelength for a period of 6 hours after induction. Split halves and splicing controls show no fluorescence. Simultaneous expression of the split parts leads to a strong increase of sfGFP fluorescence.
Figure 3: Fused proteins result in fluorescence. A: Exemplary Gating of the sample. Front Scatter depicts the size, Site Scatter Granualarity of each counted event. B: Successful reconstituion of sfGFP after 4 h
Figure 4: BL-21 (DE3) expressing non-splicing control in brightfiel channel (70ms exposure). E. Coli cells can be identified.
Figure 5: BL-21 (DE3) expressing non-splicing control in Zeiss Standard GFP channel (1 s exposure, 480nm exitation, 505nm emission). Very little florescence is observed.
Figure 6: BL-21 (DE3) expressing splicing contruct in brightfiel channel (70ms exposure). E. Coli cells can again be identified.
Figure 7: BL-21 (DE3) expressing splicing in Zeiss Standard GFP channel (1 s exposure, 480nm exitation, 505nm emission). Formation of fluorescence is observed after protein splicing has taken place.