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Part:BBa_K1362173:Experience

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)
Revision as of 15:39, 2 November 2014 by MaxW (Talk | contribs) (→‎User Reviews)


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Applications of BBa_K1362173

User Reviews

UNIQ4e706b2f6b37c123-partinfo-00000000-QINU UNIQ4e706b2f6b37c123-partinfo-00000001-QINU

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Heidelberg 2014

Split sfGFP reconstitution

This part was sucessfuly used by the Heidelberg iGEM team 2014 to reconstitute the fluorecence of split superfolder GFP by protein trans-splicing using the Npu DnaE intein. In the following the results of this experiment are shown.

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.



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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