Difference between revisions of "Part:BBa K4414011"
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==Usage and Biology== | ==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 | + | 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. |
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===Method=== | ===Method=== | ||
| − | HEK-293T cells were co-transfected with the lov2-C_inteint3- | + | 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=== | ===Result=== | ||
| − | Cells expressing both | + | 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. |
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| − | Figure1. Reconstitution of split-EGFP mediated by blue light-stimulated protein trans-splicing. (a) the lov2-C_inteint3- | + | 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
