Coding

Part:BBa_K5301013

Designed by: xuan zhang   Group: iGEM24_BNU-China   (2024-09-23)
Revision as of 05:25, 1 October 2024 by Zx1028 (Talk | contribs)


spNW15

SpNW15 is the truncated form of spNW30. spNW15 could construct nanodiscs with diameter of 15nm, and is circularized by SpyTag-Spycatcher.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 496
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 496
    Illegal NheI site found at 64
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 496
    Illegal BglII site found at 552
    Illegal BglII site found at 924
    Illegal BamHI site found at 97
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 496
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 496
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
    Illegal AgeI site found at 1027
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The theoretical molecular weight of NW15 is 40.3kDa[1], and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is around 40kDa. spNW15 protein was successfully expressed(Figure 1).
Figure 1. SDS analysis of spNW50 protein extract. Compared with the mother liquor without IPTG induction in the first column, it was obvious that proteins with required molecular weight were produced.
After SDS-PAGE, we performed Size-Exclusion Chromatography on the proteins with the aforementioned concentrations of 100 mM and 300 mM imidazole, and obtained the pure protein
Figure 1. SDS analysis of spNW50 protein extract. Compared with the mother liquor without IPTG induction in the first column, it was obvious that proteins with required molecular weight were produced.

References

[1]Zhang, S., Ren, Q., Novick, S.J. et al. One-step construction of circularized nanodiscs using SpyCatcher-SpyTag. Nat Commun 12, 5451 (2021). https://doi.org/10.1038/s41467-021-25737-7.

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