Difference between revisions of "Part:BBa K1362173"
 
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Visit the Heidelberg 2014's [http://2014.igem.org/Project wiki] for a detailed documentation or find a summary of our results in the [[{{PAGENAME}}:Experience|experience section]] of this part.
 
Visit the Heidelberg 2014's [http://2014.igem.org/Project wiki] for a detailed documentation or find a summary of our results in the [[{{PAGENAME}}:Experience|experience section]] of this part.
  
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===Usage and Biology===
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===Split sfGFP reconstitution===
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[[File:Heidelberg2014_half_sfGFP_cloningstrategy-1.PNG|400px|thumb|'''Figure 1''': <b>Cloning Strategy for the bicistronic expression of the two parts of split sfGFP.</b>
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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. ]]
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[[File:Heidelberg2014_orig_sfGFP_Platereader-6.PNG|400px|thumb|'''Figure 2''': <b>Successful ''in vivo'' restorarion of sfGFP fluorescence.</b>
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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. ]]
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[[File:Heidelberg2014_orig_FACS_sfGFP.png|400px|thumb|'''Figure 3''':  <b>Fused proteins result in fluorescence.</b>
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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 ]]
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<div style="float:right;"><gallery>
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File:Heidelberg2014_sfgfp_splicing_brightfield.png|'''Figure 4''': BL-21 (DE3) expressing non-splicing control in brightfiel channel (70ms exposure). E. Coli cells can be identified.
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File:Heidelberg2014_Sfgfp_nonsplicing_fluoro.png|'''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.
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File:Heidelberg_2014_Sfgfp_splicing_brightfield.png|'''Figure 6''': BL-21 (DE3) expressing splicing contruct in brightfiel channel (70ms exposure). E. Coli cells can again be identified.
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File:Heidelberg_2014_Sfgfp_splicing_fluoro.png|'''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.</gallery></div>
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Latest revision as of 15:19, 2 November 2014

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 [http://2014.igem.org/Project 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.



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