Difference between revisions of "Part:BBa K4414011"
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<partinfo>BBa_K4414011 short</partinfo>
 
  
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<partinfo>BBa_K4414021 short</partinfo>
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This basic part is a C-terminally 3 bases-truncated C-terminal of Npu DnaE intein (BBa_K4088893) sequence.
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==Usage and Biology==
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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
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K4414011 parameters</partinfo>
 
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==Fuctional validation==
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We constructed a plasmid that linked LBD to a C-terminal LOV2(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.
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===Method===
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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.
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===Result===
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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 BBa_K4088893 to BBa_K4414011 enabled lov2-mediated light-inducible protein splicing in mammalian cells.
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<figure class="figure">
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<img src="https://static.igem.wiki/teams/4414/wiki/011.png" class="figure-img img-fluid rounded"  height="230px">
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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.
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===Reference===
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1. Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nature reviews. Molecular cell biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152

Revision as of 16:37, 11 October 2022


LBD-EGFP

This basic part is a C-terminally 3 bases-truncated C-terminal of Npu DnaE intein (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


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(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 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.

Reference

1. Weikum, E. R., Knuesel, M. T., Ortlund, E. A., & Yamamoto, K. R. (2017). Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nature reviews. Molecular cell biology, 18(3), 159–174. https://doi.org/10.1038/nrm.2016.152