Difference between revisions of "Part:BBa K5301015"
Line 24: Line 24:
 
<h2>Characterization</h2>
 
<h2>Characterization</h2>
  
−
<h3>Functional Verification</h3>
 
 
The theoretical molecular weight of NW50 is 123.9kDa[1], and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is 150kDa.
 
The theoretical molecular weight of NW50 is 123.9kDa[1], and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is 150kDa.
 
Due to the large molecular weight of the protein, NW50 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 1). A more specific exploration of protein dimerization conditions can be found in the engineering section of 2024BNUChina iGEM.
 
Due to the large molecular weight of the protein, NW50 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 1). A more specific exploration of protein dimerization conditions can be found in the engineering section of 2024BNUChina iGEM.
  
−
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">
+
<div class="center">
−
https://static.igem.wiki/teams/5301/parts/sds-results-of-nw50-dimerization-or-monomerization.png
+
    <div class="thumb tnone">
−
</div><div class="thumbcaption">Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).
+
        <div class="thumbinner" style="width:50%;">
−
</div></div></div></div>
+
            <a href="https://static.igem.wiki/teams/4634/wiki/parts-registry/bba-k4634000-2.jpg" class="image">
 +
                <img alt="" src="https://static.igem.wiki/teams/5301/parts/sds-results-of-nw50-dimerization-or-monomerization.png" width="100%" height=auto class="thumbimage" /></a>                 <div class="thumbcaption">
 +
                <div class="magnify">
 +
                    <a href="https://static.igem.wiki/teams/4634/wiki/parts-registry/bba-k4634000-2.jpg" class="internal" title="Enlarge"></a>
 +
                </div>
 +
                <b>Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).</b>
 +
               
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>
  
 
<h2>Conclusion</h2>
 
<h2>Conclusion</h2>
 
According to the results of electron microscopy and DLS, NW50 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 100nm(Figure 2).
 
According to the results of electron microscopy and DLS, NW50 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 100nm(Figure 2).
−
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:0.5;overflow:hidden;">https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph.jpg</div><div class="thumbcaption">Figure 2. Electron microscopic images of nanodiscs with a particle size of about 100nm prepared by NW50. The particle size is in agreement with our DLS results.</div></div></div></div>
+
 
 +
<div class="center">
 +
    <div class="thumb tnone">
 +
        <div class="thumbinner" style="width:50%;">
 +
            <a href="https://static.igem.wiki/teams/4634/wiki/parts-registry/bba-k4634000-2.jpg" class="image">
 +
                <img alt="" src="https://static.igem.wiki/teams/5301/parts/nw50-electron-microscope-photograph.jpg" width="100%" height=auto class="thumbimage" /></a>                 <div class="thumbcaption">
 +
                <div class="magnify">
 +
                    <a href="https://static.igem.wiki/teams/4634/wiki/parts-registry/bba-k4634000-2.jpg" class="internal" title="Enlarge"></a>
 +
                </div>
 +
                <b>Figure 2. Electron microscopic images of nanodiscs with a particle size of about 100nm prepared by NW50. The particle size is in agreement with our DLS results.</b>
 +
               
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>
  
 
<h2>References</h2>
 
<h2>References</h2>
 
[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.
 
[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.

Revision as of 08:58, 30 September 2024


spNW50 is high molecular weight membrane scaffold protein used to produce large nanodiscs.

NW50 is high molecular weight membrane scaffold protein used to produce large nanodiscs.spNW50 could construct nanodiscs with large diameter, and is circularized by SpyTag-Spycatcher.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 1576
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
    Illegal PstI site found at 1204
    Illegal PstI site found at 1237
    Illegal PstI site found at 1744
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 1576
    Illegal NheI site found at 64
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
    Illegal PstI site found at 1204
    Illegal PstI site found at 1237
    Illegal PstI site found at 1744
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 1576
    Illegal BglII site found at 924
    Illegal BglII site found at 1464
    Illegal BglII site found at 1632
    Illegal BamHI site found at 97
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 1576
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
    Illegal PstI site found at 1204
    Illegal PstI site found at 1237
    Illegal PstI site found at 1744
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 496
    Illegal EcoRI site found at 1576
    Illegal SpeI site found at 448
    Illegal PstI site found at 664
    Illegal PstI site found at 1204
    Illegal PstI site found at 1237
    Illegal PstI site found at 1744
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

The theoretical molecular weight of NW50 is 123.9kDa[1], and the actual molecular weight size verified by SDS-PAGE in literature and practical experiments is 150kDa. Due to the large molecular weight of the protein, NW50 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 1). A more specific exploration of protein dimerization conditions can be found in the engineering section of 2024BNUChina iGEM.
Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).

Conclusion

According to the results of electron microscopy and DLS, NW50 and lipid DOPC can be successfully used to fabricate nanodiscs with a particle size of about 100nm(Figure 2).
Figure 2. Electron microscopic images of nanodiscs with a particle size of about 100nm prepared by NW50. The particle size is in agreement with our DLS results.

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.