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 and used it in level 1 biological laboratory.

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Revision as of 14:53, 13 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 and used it in level 1 biological laboratory.

Sequence and Features

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_71-239 expressing and the EGFP_1-70-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 EGFP_1-70-N_intein and the improved lov2-C_inteint3-EGFP_71-239 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-EGFP_71-239 expressing and the EGFP_1-70-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-EGFP_71-239 expressing and the EGFP_1-70-N_intein expressing cells w/wo blue light treatment.