Difference between revisions of "Part:BBa K4414011"
 
(5 intermediate revisions by 2 users not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 +
 
<partinfo>BBa_K4414011 short</partinfo>
 
<partinfo>BBa_K4414011 short</partinfo>
  
−
Blank
+
This basic part is a C-terminally 3 bases-truncated C-terminal of Npu DnaE intein ([[Part:BBa_K4088893]]) sequence.
 +
 
 +
==Usage and Biology==
 +
 
 +
Intein is a segment of a protein that can self-catalytically excise out and ligate the remaining parts of the protein (N- and C-exteins) with a peptide bond. For this part, we used the Npu DnaE intein. DnaE - alpha subunit of the DNA polymerase III intein. This intein is identified as a naturally occurring split intein in Nostoc punctiforme. We obtained this DNA by DNA synthesis.This part can be used in level 1 biological laboratory.
 +
 
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->
Line 17: Line 20:
 
<partinfo>BBa_K4414011 parameters</partinfo>
 
<partinfo>BBa_K4414011 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
==Fuctional validation==
 +
We constructed a plasmid that linked LBD to a C-terminal LOV2([[Part:BBa_K4016004]]) domain, and a C-terminal truncated EGFP retaining 71th to 239th base to verify the function of C_Inteint3. It is designed to respond to the regulation of blue light and fuse the isolated EGFP.
 +
===Method===
 +
 +
HEK-293T cells were co-transfected with the lov2-C_inteint3-EGFP<sub>71-239</sub> expressing and the EGFP<sub>1-70</sub>-N_intein expressing plasmids. Cells were either exposed to blue light (with ON [4 s]-OFF [56 s] cycles) or maintained under dark conditions for control since the 6th hour post-transfection. Cells were imaged or harvested for Western Blotting 48 h post-light illumination.
 +
 +
===Result===
 +
 +
Cells expressing both EGFP<sub>1-70</sub>-N_intein and the improved lov2-C_inteint3-EGFP<sub>71-239</sub> proteins showed nearly no fluorescent signal in control cells that were not illuminated by blue light, and a strong GFP signal in blue light treated group (Figure 1a). The reconstitution of EGFP was also validated via Western Blotting, which showed a significantly stronger EGFP signal in blue-light treated cells compared to the untreated cells (Figure 1b). These results demonstrated that the improvement from ([[Part:BBa_K4088893]]) to BBa_K4414011 enabled lov2-mediated light-inducible protein splicing in mammalian cells.
 +
 +
<html>
 +
 +
<figure class="figure">
 +
<img src="https://static.igem.wiki/teams/4414/wiki/011.png" class="figure-img img-fluid rounded"  height="400px">
 +
 +
</figure>
 +
 +
</html>
 +
Figure1. Reconstitution of split-EGFP mediated by blue light-stimulated protein trans-splicing. (a) the lov2-C_inteint3-EGFP<sub>71-239</sub> expressing and the EGFP<sub>1-70</sub>-N_intein expressing plasmids in HEK-293 cells exposed in dark(left) or in blue light(right); (b) Immunoblotting of the indicated proteins in the lov2-C_inteint3-EGFP<sub>71-239</sub> expressing and the EGFP<sub>1-70</sub>-N_intein expressing cells w/wo blue light treatment.

Latest revision as of 03:54, 14 October 2022


C-intein_t3

This basic part is a C-terminally 3 bases-truncated C-terminal of Npu DnaE intein (Part:BBa_K4088893) sequence.

Usage and Biology

Intein is a segment of a protein that can self-catalytically excise out and ligate the remaining parts of the protein (N- and C-exteins) with a peptide bond. For this part, we used the Npu DnaE intein. DnaE - alpha subunit of the DNA polymerase III intein. This intein is identified as a naturally occurring split intein in Nostoc punctiforme. We obtained this DNA by DNA synthesis.This part can be used in level 1 biological laboratory.


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
    COMPATIBLE WITH RFC[1000]


Fuctional validation

We constructed a plasmid that linked LBD to a C-terminal LOV2(Part:BBa_K4016004) domain, and a C-terminal truncated EGFP retaining 71th to 239th base to verify the function of C_Inteint3. It is designed to respond to the regulation of blue light and fuse the isolated EGFP.

Method

HEK-293T cells were co-transfected with the lov2-C_inteint3-EGFP71-239 expressing and the EGFP1-70-N_intein expressing plasmids. Cells were either exposed to blue light (with ON [4 s]-OFF [56 s] cycles) or maintained under dark conditions for control since the 6th hour post-transfection. Cells were imaged or harvested for Western Blotting 48 h post-light illumination.

Result

Cells expressing both EGFP1-70-N_intein and the improved lov2-C_inteint3-EGFP71-239 proteins showed nearly no fluorescent signal in control cells that were not illuminated by blue light, and a strong GFP signal in blue light treated group (Figure 1a). The reconstitution of EGFP was also validated via Western Blotting, which showed a significantly stronger EGFP signal in blue-light treated cells compared to the untreated cells (Figure 1b). These results demonstrated that the improvement from (Part:BBa_K4088893) to BBa_K4414011 enabled lov2-mediated light-inducible protein splicing in mammalian cells.

Figure1. Reconstitution of split-EGFP mediated by blue light-stimulated protein trans-splicing. (a) the lov2-C_inteint3-EGFP71-239 expressing and the EGFP1-70-N_intein expressing plasmids in HEK-293 cells exposed in dark(left) or in blue light(right); (b) Immunoblotting of the indicated proteins in the lov2-C_inteint3-EGFP71-239 expressing and the EGFP1-70-N_intein expressing cells w/wo blue light treatment.