Difference between revisions of "Part:BBa K5461000"

 
 
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<partinfo>BBa_K5461000 short</partinfo>
 
<partinfo>BBa_K5461000 short</partinfo>
  
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CsgA is a biofilm protein that does not assemble within E. coli but can self-assemble into fibrous structures when expressed in vitro. This part is designed to express a protein with a GST purification tag and a SpyTag oligopeptide, under the control of the T7 promoter.
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We have expressed the fusion protein csgA-SpyTag with a GST tag within E. coli and successfully purified it using column chromatography.
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In our system, the csgA-SpyTag fusion protein serves as a linker between cellulose and the target protein.
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    <div style="text-align: left;">
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        <p><strong>GST-csga-spytag</strong><br><strong>Description:</strong><br>This part can be used to express Csga tagged with both a GST tag and SpyTag.</p>
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        <div style="display: flex; justify-content: center; align-items: center;">
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            <img src="https://static.igem.wiki/teams/5461/part-registry/plasmid-curlis1.jpg" alt="图1" style="width: 400px; margin-right: 10px;">
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        </div>
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        <p>Csga has been shown to self-assemble into curli fibers and bind to cellulose, while SpyTag has been proven to tightly bind to SpyCatcher through an isopeptide bond. Thus, this construct can serve as a scaffold protein, acting as <strong>moduleII</strong> of our system. On one hand, it binds to cellulose, and on the other, it binds to SpyCatcher-tagged proteins. Therefore, using this part in conjunction with BBa_K5461000 enables the coupling of more functional proteins to cellulose, facilitating cellulose functionalization:</p>
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<div style="display: flex; justify-content: center; align-items: center;">
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            <img src="https://static.igem.wiki/teams/5461/part-registry/module.jpg" alt="图2" style="width: 400px; margin-right: 10px;">
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        </div>
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<p><strong>Results:</strong><br>The biofilm protein Csga can be stained with Congo red, so we verified the expression of the Csga membrane protein using a Congo red agar plate assay. Following that, through liquid culture expression experiments, we successfully produced and collected filamentous precipitates: </p>
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  <div style="display: flex; justify-content: center; align-items: center;">
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    <img src="https://static.igem.wiki/teams/5461/part-registry/congo-red-letf.jpg" alt="图3" style="width: 225px; margin-right: 10px;">
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            <img src="https://static.igem.wiki/teams/5461/part-registry/congo-red-right.jpg" alt="图4" style="width: 235px; margin-right: 10px;">
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  <img src="https://static.igem.wiki/teams/5461/part-registry/fermentation-of-curlis-left.jpg" alt="图5" style="width: 195px; margin-right: 10px;">
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          <img src="https://static.igem.wiki/teams/5461/part-registry/fermentation-of-curlis-right.jpg" alt="图6" style="width: 225px; margin-right: 10px;">
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      </div>
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<p>After purification of Csga using a GST affinity chromatography column, the protein was further analyzed by Western blot. Protein samples were separated on a SDS-PAGE gel, transferred onto a PVDF membrane, and probed with an anti-GST antibody. The presence of Csga-GST was confirmed by the detection of the expected band size, indicating successful purification and expression</p>
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        <div style="display: flex; justify-content: center; align-items: center;">
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    <img src="https://static.igem.wiki/teams/5461/part-registry/curliswb.jpg" alt="图7" style="width: 280px; margin-right: 10px;">
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        </div>
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      <p>Next, we purified the colored protein using BBa_K5461001 as our proof-of-concept protein. After obtaining the purified protein, we validated the binding interactions between bacterial cellulose, the Curlis-SpyTag scaffold protein, and the SpyCatcher-POI. The results demonstrated that the scaffold protein system significantly enhances the binding efficiency between the target protein (POI) and cellulose.</p>
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  <div style="display: flex; justify-content: center; align-items: center;">
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      <img src="https://static.igem.wiki/teams/5461/part-registry/sfgfp-combination.jpg" alt="图8" style="width: 325px; margin-right: 10px;">
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          <img src="https://static.igem.wiki/teams/5461/part-registry/amilcp-combination.jpg" alt="图9" style="width: 325px; margin-right: 10px;">
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        </div>
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    <p>Through this demonstration, we successfully validated the efficiency of the Curlis-Spy scaffold protein design and the plug-and-play nature of the system. In the future, we plan to provide more protein data.</p><br><p><strong>Summary:</strong><br>We have established a standard, offering a foundational and scalable platform that decouples protein functionality from its compatibility with cellulose. This allows future iGEM teams to focus on selecting proteins for cellulose modification without needing to worry about their binding capability to cellulose, providing a foundational framework for the development of functionalized cellulose.<p>
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    </div>
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</body>
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</html>
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=== Reference ===
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[1] https://2014.igem.org/Team:Imperial/Project_Background<br>
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[2] https://2017.igem.org/Team:TUST_China/Description<br>
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[3] https://2020.igem.org/Team:JNFLS/Design<br>
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[4] https://2021.igem.org/Team:SZPT-CHINA/Design<br>
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[5] Khatri, V., Jafari, M., Gaudreault, R., et al., 2023. Bionanocomposites with enhanced physical properties from curli amyloid assemblies and cellulose nanofibrils. Biomacromolecules, 24(11), pp.5290-5302. doi:10.1021/acs.biomac.3c00786.<br>
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[6] Hatlem, D., Trunk, T., Linke, D. & Leo, J.C., 2019. Catching a SPY: using the SpyCatcher-SpyTag and related systems for labeling and localizing bacterial proteins. International Journal of Molecular Sciences, 20(9), p.2129. Published on 30 April 2019. doi:10.3390/ijms20092129.<br>
  
 
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===Usage and Biology===
 
===Usage and Biology===
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This part is capable of expressing a fusion protein comprising CSGA-SpyTag with a GST protein tag. The GST tag facilitates protein purification, while CSGA has been demonstrated to tightly bind to cellulose and enhance its mechanical properties. Additionally, SpyTag can covalently bind to SpyCatcher. This architectural design allows the fusion protein to serve as a scaffold for modifying cellulose. In our project, we express SpyCatcher and the protein of interest (POI) as a fusion protein, which is then combined with cellulose, thereby endowing cellulose with the properties of various functional proteins.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 11:20, 1 October 2024


GST-csgA-spytag

GST-csga-spytag
Description:
This part can be used to express Csga tagged with both a GST tag and SpyTag.

图1

Csga has been shown to self-assemble into curli fibers and bind to cellulose, while SpyTag has been proven to tightly bind to SpyCatcher through an isopeptide bond. Thus, this construct can serve as a scaffold protein, acting as moduleII of our system. On one hand, it binds to cellulose, and on the other, it binds to SpyCatcher-tagged proteins. Therefore, using this part in conjunction with BBa_K5461000 enables the coupling of more functional proteins to cellulose, facilitating cellulose functionalization:

图2

Results:
The biofilm protein Csga can be stained with Congo red, so we verified the expression of the Csga membrane protein using a Congo red agar plate assay. Following that, through liquid culture expression experiments, we successfully produced and collected filamentous precipitates:

图3 图4 图5 图6

After purification of Csga using a GST affinity chromatography column, the protein was further analyzed by Western blot. Protein samples were separated on a SDS-PAGE gel, transferred onto a PVDF membrane, and probed with an anti-GST antibody. The presence of Csga-GST was confirmed by the detection of the expected band size, indicating successful purification and expression

图7

Next, we purified the colored protein using BBa_K5461001 as our proof-of-concept protein. After obtaining the purified protein, we validated the binding interactions between bacterial cellulose, the Curlis-SpyTag scaffold protein, and the SpyCatcher-POI. The results demonstrated that the scaffold protein system significantly enhances the binding efficiency between the target protein (POI) and cellulose.

图8 图9

Through this demonstration, we successfully validated the efficiency of the Curlis-Spy scaffold protein design and the plug-and-play nature of the system. In the future, we plan to provide more protein data.


Summary:
We have established a standard, offering a foundational and scalable platform that decouples protein functionality from its compatibility with cellulose. This allows future iGEM teams to focus on selecting proteins for cellulose modification without needing to worry about their binding capability to cellulose, providing a foundational framework for the development of functionalized cellulose.

Reference

[1] https://2014.igem.org/Team:Imperial/Project_Background
[2] https://2017.igem.org/Team:TUST_China/Description
[3] https://2020.igem.org/Team:JNFLS/Design
[4] https://2021.igem.org/Team:SZPT-CHINA/Design
[5] Khatri, V., Jafari, M., Gaudreault, R., et al., 2023. Bionanocomposites with enhanced physical properties from curli amyloid assemblies and cellulose nanofibrils. Biomacromolecules, 24(11), pp.5290-5302. doi:10.1021/acs.biomac.3c00786.
[6] Hatlem, D., Trunk, T., Linke, D. & Leo, J.C., 2019. Catching a SPY: using the SpyCatcher-SpyTag and related systems for labeling and localizing bacterial proteins. International Journal of Molecular Sciences, 20(9), p.2129. Published on 30 April 2019. doi:10.3390/ijms20092129.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 85