Difference between revisions of "Part:BBa K5301015"
| Line 4: | Line 4: | ||
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. | 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. | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
| Line 18: | Line 9: | ||
<!-- --> | <!-- --> | ||
| − | + | ||
| + | ===Sequence and Features=== | ||
| + | <span class='h3bb'></span> | ||
<partinfo>BBa_K5301015 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5301015 SequenceAndFeatures</partinfo> | ||
| Line 26: | Line 19: | ||
<partinfo>BBa_K5301015 parameters</partinfo> | <partinfo>BBa_K5301015 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| + | |||
| + | <html> | ||
| + | |||
| + | <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. | ||
| + | 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;">https://static.igem.wiki/teams/5301/parts/sds-results-of-nw50-dimerization-or-monomerization.png</div><div class="thumbcaption">Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).</div></div></div></div> | ||
| + | |||
| + | <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). | ||
| + | <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> | ||
| + | |||
| + | <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. | ||
Revision as of 08:43, 30 September 2024
NW50 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:
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
Characterization
Functional Verification
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.