Difference between revisions of "Part:BBa K5301003"

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<partinfo>BBa_K5301003 parameters</partinfo>
 
<partinfo>BBa_K5301003 parameters</partinfo>
 
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<h2>Characterization</h2>
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In the design, we intend to use the robust reaction conditions and irreversible connections of SpyCatcher and SpyTag to link the two ends of the MSP protein, thereby enabling the formation of nanodisks.
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In the experiment, we successfully characterized the NW50 [BBa_K5301015] protein with spytag and spycatcher, but their presence promoted the dimerization of the protein (Figure 1a). Through the engineering of iGEM24_BNU-China, we alleviated the dimerization problem and produced a monomeric protein with two tags (Figure 1bc).
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                <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">
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                <b>Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).</b>
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<h2>Conclusion</h2>
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Finally, through electron microscope imaging, we determined that the nanodisk was successfully generated under the action of spytag and spycatcher(Figure 2).
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                <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">
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                <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>
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Revision as of 12:47, 30 September 2024


SpyTag can achieve covalent binding of proteins through Tag-Catcher interaction.

SpyTag comes from the spontaneous isopeptide bond domain in streptococcus pyogenes fibronectin-binding protein FbaB. It can cooperate with SpyCatcher to achieve covalent binding of proteins through Tag-Catcher interaction. The robust reaction conditions and irreversible linkage of SpyTag-Catcher provide a targetable lock in cells and a stable module for new protein architectures.

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]


Characterization

In the design, we intend to use the robust reaction conditions and irreversible connections of SpyCatcher and SpyTag to link the two ends of the MSP protein, thereby enabling the formation of nanodisks. In the experiment, we successfully characterized the NW50 [BBa_K5301015] protein with spytag and spycatcher, but their presence promoted the dimerization of the protein (Figure 1a). Through the engineering of iGEM24_BNU-China, we alleviated the dimerization problem and produced a monomeric protein with two tags (Figure 1bc). 

           <a href="sds-results-of-nw50-dimerization-or-monomerization.png" class="image">
<img alt="" src="sds-results-of-nw50-dimerization-or-monomerization.png" width="100%" height=auto class="thumbimage" /></a> 
                   <a href="sds-results-of-nw50-dimerization-or-monomerization.png" class="internal" title="Enlarge"></a>

               Figure 1. SDS analysis of NW50 results of dimerization(a) and monomerization(bc).

Conclusion

Finally, through electron microscope imaging, we determined that the nanodisk was successfully generated under the action of spytag and spycatcher(Figure 2). 

           <a href="nw50-electron-microscope-photograph.jpg" class="image">
<img alt="" src="nw50-electron-microscope-photograph.jpg" width="100%" height=auto class="thumbimage" /></a> 
                   <a href="nw50-electron-microscope-photograph.jpg" class="internal" title="Enlarge"></a>

               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.