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Translational_Unit

Part:BBa_K1362173

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)

Split sfGFP reconstitution N-terminal splicing half

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



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 66


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