Coding

Part:BBa_K5301022

Designed by: Jiaxin Cao   Group: iGEM24_BNU-China   (2024-09-24)
Revision as of 04:34, 1 October 2024 by Caojiaxin (Talk | contribs)


SpyCatcher-spNW50-SpyTag achieving self-cyclization of MSP nanodisc.

This pathway achieving self-cyclization of NW50 to construct nanodiscs with larger particle sizes.The following figure shows the structure of the composite part(Figure 1). 

           <a href="spytag-nw50-spycatcher.png" class="image">
<img alt="" src="spytag-nw50-spycatcher.png" width="100%" height=auto class="thumbimage" /></a> 
                   <a href="spytag-nw50-spycatcher.png" class="internal" title="Enlarge"></a>

               Figure 1. Structure diagram of Spytag-NW50-Spycatcher.
               

           <a href="before-and-after-induction-of-nw50-iptg.png" class="image">
<img alt="" src="before-and-after-induction-of-nw50-iptg.png" width="100%" height=auto class="thumbimage" /></a> 
                   <a href="before-and-after-induction-of-nw50-iptg.png" class="internal" title="Enlarge"></a>

               Figure 2. SDS analysis of Spycatcher-NW50-SpyTag 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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 565
    Illegal EcoRI site found at 1645
    Illegal SpeI site found at 517
    Illegal PstI site found at 733
    Illegal PstI site found at 1273
    Illegal PstI site found at 1306
    Illegal PstI site found at 1813
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 565
    Illegal EcoRI site found at 1645
    Illegal NheI site found at 133
    Illegal NheI site found at 2061
    Illegal SpeI site found at 517
    Illegal PstI site found at 733
    Illegal PstI site found at 1273
    Illegal PstI site found at 1306
    Illegal PstI site found at 1813
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 565
    Illegal EcoRI site found at 1645
    Illegal BglII site found at 993
    Illegal BglII site found at 1533
    Illegal BglII site found at 1701
    Illegal BamHI site found at 166
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 565
    Illegal EcoRI site found at 1645
    Illegal SpeI site found at 517
    Illegal PstI site found at 733
    Illegal PstI site found at 1273
    Illegal PstI site found at 1306
    Illegal PstI site found at 1813
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 565
    Illegal EcoRI site found at 1645
    Illegal SpeI site found at 517
    Illegal PstI site found at 733
    Illegal PstI site found at 1273
    Illegal PstI site found at 1306
    Illegal PstI site found at 1813
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The total molecular weight of the composite component of Spycatcher-NW50-SpyTag is about 150kDa, and it can be seen that it can be expressed normally after IPTG induction (Figure 2).
Figure 2. SDS analysis of Spycatcher-NW50-SpyTag 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.
Due to the large molecular weight of the protein and the combined action of spytag-spycatcher, Spycatcher-NW50-SpyTag is prone to dimerization during actual protein extraction. Protein dimerization can be minimized by adding the detergent Triton X-100 to the refined extraction buffer and reducing the nickel column elution time during the refined extraction process. It should be used as soon as possible after protein extraction to prevent the protein from self-dimerizing after a period of time(Figure 3). A more specific exploration of protein dimerization conditions can be found in the engineering section of iGEM24_BNU-China.
Figure 3. SDS analysis of Spycatcher-NW50-SpyTag results of dimerization(a) and monomerization(bc).

Conclusion

According to the results of electron microscopy and DLS, Spycatcher-NW50-SpyTag and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 200-300nm(Figure 4).
Figure 4. Electron microscopy (a) and DLS results (b) of nanodiscs produced by Spycatcher-NW50-SpyTag. It can be seen that the two results are consistent, and the size of the nanodisc is about 200-300nm.

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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