Coding

Part:BBa_K4088893

Designed by: Ruslan Vasiliev, Yaroslav Ugolkov, Alice Potapova, Pavel Ivanov-Rostovtsev   Group: iGEM21_Moscow_City   (2021-10-21)


DnaE - C

C-terminal of Npu DnaE intein.

You can find N-terminal of Npu DnaE intein there - BBa_K4088892.

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]





Usage and Biology

Intein
Intein is a segment of a protein that can self-catalytically excised out and ligate the remaining parts of the protein (N- and C-exteins) with a peptide bond [1].
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 [2]. Its first amino acid is cysteine which is important for protein trans-splicing to take place.

This part we used in the сomposite system - С-terminal fragment of β-lactamase fused with dCas13a and C-terminal intein (BBa_K4088890).


Improvement:NUDT_CHINA 2022

Our improvement is C-intein-t3 (Part:BBa_K4414011) a C-terminally 3 bases-truncated C-terminal of Npu DnaE intein (Part:BBa_K4088893) sequence. 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. The improved C-intein-t3 shares a similar nucleic acid sequence to the original DnaE – C in BBa_K4088893, with only 3 bases at the c-terminus truncated. However, these differences enabled Lov2-mediated blue-light control of protein splicing.

Methods

We evaluated whether the improved C_intein can mediate the lov2-based blue light responsiveness by assaying the intein-mediated reconstitution of split-EGFP. Hence, we first fused the lov2 domain (Part:BBa_K4016004) and the EGFP71-239 domain on the N-terminus and the C-terminus of C_inteint3 respectively. A similar construct using the original C_intein was also constructed as a head-to-head control. Also, another plasmid expressing the fusion protein containing the N_Intein (Part:BBa_K4088892) and the EGFP1-70 domain was obtained.

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


Results

The fluorescent imaging of the cells expressing both lov2-C_intein-EGFP71-239 and EGFP1-70-N_intein proteins showed strong EGFP signal in both the blue-light-illuminated group and control group kept under dark conditions. Suggesting that lov2 is not capable of inhibiting the function of the original C_intein (BBa_K4088893) under dark conditions. Intriguingly, 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 (Part: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) Top: the lov2-C_intein-EGFP71-239 expressing and the EGFP1-70-N_intein expressing plasmids in HEK-293 cells exposed in dark(left) or in blue light(right); Button: 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. (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.

References

  1. ↑ Anraku, Y., Mizutani, R. and Satow, Y. (2005), Protein Splicing: Its Discovery and Structural Insight into Novel Chemical Mechanisms. IUBMB Life, 57: 563-574. https://doi.org/10.1080/15216540500215499
  2. ↑ Oeemig JS, Aranko AS, Djupsjöbacka J, Heinämäki K, Iwaï H. Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification. FEBS Lett. 2009 May 6;583(9):1451-6. https://doi.org/10.1016/j.febslet.2009.03.058
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