Difference between revisions of "Part:BBa K5461001"
 
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<partinfo>BBa_K5461001 short</partinfo>
 
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The New Composite Part BBa_K5461001 functions to form a POI-SpyCatcher fusion protein for linking with the SpyTag, serving as the "plug-and-play functional module" of the bacterial cellulose modification machine. The structure of BBa_K5461001 is J23119-BBa_B0034-POI-SpyCatcher-BBa_B1006. We utilized commonly used iGEM promoter J23119, RBS BBa_B0034, and terminator BBa_B1006. Additionally, we designed standard BsaI interfaces (GGTCTCaGGTAtctagt - POI - GAGCtGAGACCa) on both sides of the POI, allowing for easy replacement of the POI. The POI standard interface in BBa_K5461000 implies the infinite potential for extending the functionalities of the bacterial cellulose modification machine. Future iGEMers can utilize this system to attempt various modifications of bacterial cellulose or even perform high-throughput screening by replacing the POI with a POI library.
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        <p><strong>Plug-and-Play Protein Assembly Module</strong><br><strong>Description:</strong><br>This part enables us to produce proteins with functional SpyCatcher tags, which can bind to the scaffold proteins produced by BBa_K5461000, thereby enhancing the binding efficiency between the protein of interest (POI) and bacterial 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/plasmid-poi.jpg" alt="图一" style="width: 600px; margin-right: 10px;">
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        </div>
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        <p>We used the common iGEM promoter J23119, RBS BBa_B0034, terminator BBa_B1006, and a SpyCatcher. Additionally, we designed a standard BsaI interface upstream of the SpyCatcher (GGTAAGAGACC - POI - GGTCTCATACCa), making it easy to replace the POI and generate various functional proteins that can be used for bacterial cellulose modification. Future iGEM teams can leverage this system to attempt various modifications to bacterial cellulose, and they could even perform high-throughput screening by replacing a library of POIs.</p>
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<p><strong>Results:</strong><br>In this project, we utilized two colored proteins, sfGFP and amilCP, to complete the proof of concept (POC). </p>
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    <img src="https://static.igem.wiki/teams/5461/part-registry/gfp-plasmid.jpg" alt="图2" style="width: 400px; margin-right: 10px;">
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<div style="margin: 0 30px;"></div>
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            <img src="https://static.igem.wiki/teams/5461/part-registry/amilcp-plasmid.jpg" alt="图3" style="width: 400px; margin-right: 10px;">
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<p>We successfully expressed the SpyCatcher-tagged sfGFP and amilCP fluorescent proteins and confirmed their expression and purification through SDS-PAGE molecular weight comparison and Western Blot (WB) analysis.</p>
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    <img src="https://static.igem.wiki/teams/5461/part-registry/fp200.jpg" alt="图2" style="width: 220px; margin-right: 10px;">
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    <div style="width: 1px; height: 200px; background-color: black; margin: 0 15px;"></div>
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    <img src="https://static.igem.wiki/teams/5461/part-registry/fp-wb.jpg" alt="图1" style="width: 450px;">
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      <p>Next, we purified Curlis-SpyTag using the BBa_K5461000. After obtaining the purified Curlis-SpyTag, we validated the binding 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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      <img src="https://static.igem.wiki/teams/5461/part-registry/sfgfp-combination.jpg" alt="图2" style="width: 380px; margin-right: 10px;">
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          <img src="https://static.igem.wiki/teams/5461/part-registry/amilcp-combination.jpg" alt="图2" style="width: 380px; margin-right: 10px;">
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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><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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=== 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>
  
 
<!-- Add more about the biology of this part here
 
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Latest revision as of 11:13, 1 October 2024


J23119-BBa_B0034- POI -Spycatcher- BBa_B1006

Plug-and-Play Protein Assembly Module
Description:
This part enables us to produce proteins with functional SpyCatcher tags, which can bind to the scaffold proteins produced by BBa_K5461000, thereby enhancing the binding efficiency between the protein of interest (POI) and bacterial cellulose.

图一

We used the common iGEM promoter J23119, RBS BBa_B0034, terminator BBa_B1006, and a SpyCatcher. Additionally, we designed a standard BsaI interface upstream of the SpyCatcher (GGTAAGAGACC - POI - GGTCTCATACCa), making it easy to replace the POI and generate various functional proteins that can be used for bacterial cellulose modification. Future iGEM teams can leverage this system to attempt various modifications to bacterial cellulose, and they could even perform high-throughput screening by replacing a library of POIs.

Results:
In this project, we utilized two colored proteins, sfGFP and amilCP, to complete the proof of concept (POC).

图2
图3

We successfully expressed the SpyCatcher-tagged sfGFP and amilCP fluorescent proteins and confirmed their expression and purification through SDS-PAGE molecular weight comparison and Western Blot (WB) analysis.

图2
图1

Next, we purified Curlis-SpyTag using the BBa_K5461000. After obtaining the purified Curlis-SpyTag, we validated the binding 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2 图2

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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]