Difference between revisions of "Part:BBa K1362173:Experience"

 
(→‎User Reviews)
Line 19: Line 19:
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
 
<!-- DON'T DELETE --><partinfo>BBa_K1362173 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K1362173 EndReviews</partinfo>
 +
 +
{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
 +
<partinfo>BBa_K1362173 AddReview 5</partinfo>
 +
<I>Heidelberg 2014</I>
 +
|width='60%' valign='top'|
 +
 +
===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.
 +
 +
[[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>
 +
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. ]]
 +
[[File:Heidelberg2014_orig_sfGFP_Platereader-6.PNG|400px|thumb|'''Figure 2''': <b>Successful ''in vivo'' restorarion of sfGFP fluorescence.</b>
 +
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. ]]
 +
[[File:Heidelberg2014_orig_FACS_sfGFP.png|400px|thumb|'''Figure 3''':  <b>Fused proteins result in fluorescence.</b>
 +
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 ]]
 +
<div style="float:right;"><gallery>
 +
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.
 +
 +
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.
 +
 +
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.
 +
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>
 +
 +
 +
 +
 +
<!-- -->
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K1362173 SequenceAndFeatures</partinfo>
 +
 +
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_K1362173 parameters</partinfo>
 +
<!-- -->
 +
 +
|};

Revision as of 15:39, 2 November 2014


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1362173

User Reviews

UNIQ5fff53b859662a6e-partinfo-00000000-QINU UNIQ5fff53b859662a6e-partinfo-00000001-QINU

•••••

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


;