Difference between revisions of "Part:BBa K2132001"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2132001 short</partinfo>
 
<partinfo>BBa_K2132001 short</partinfo>
  
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This is the subunit of the biofilm of E. Coli Curli system linked to SpyCatcher and HisTag. The two added parts can be used for various functions with SpyTag and other His tag-binding materials like quantum dots.
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This is the subunit of major proteins of E. Coli biofilm Curli system linked to SpyCatcher and HisTag. The two added protein tag and domain can bring new functions to biofilms . For example, SpyTag can covalently interact with any fusion protein containing Spytcatcher,  While His tag can facilitate purification, and endow biofilms with specific binding ability towards NTA-decorated nanoparticles, as demonstrated below
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<h3>Characterization</h3>
 
<h3>Characterization</h3>
 
<p>
 
<p>
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Three different experiments were conducted to characterize these biobricks:
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Four different experiments were conducted to characterize this biobrick:
 
<ul>
 
<ul>
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<li> TEM: visualization of CsgASpyCatcherHisTag mutant biofilm </li>
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<li> TEM: visualization of His-CsgA-SpyCacher-histag mutant biofilm </li>
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<li> Fluoresence Binding Test </li>
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<li> QDs’ Fluorescence Binding Test </li>
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<li> TEM: visualization of binding test </li></ul>
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<li> TEM: visualization of binding test with AuNPs </li>
 +
<li> Spy System Functional Test </li></ul>
 
</p>
 
</p>
  
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<h4> TEM: visualization of CsgASpyCatcherHisTag mutant biofilm </h4>
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<h4> TEM: visualization of His-CsgA-SpyCacher-histag mutant biofilm </h4>
  
 
<figure align="center">
 
<figure align="center">
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<img src="https://static.igem.org/mediawiki/parts/c/c9/Shanghaitechchina_CsgASpyCatcherHistag2222.png" width="50%" height="50%">
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<img src="https://static.igem.org/mediawiki/parts/7/7d/Shanghaitechchina_hsch_part.png" width="50%" height="50%">
 
<figcaption>
 
<figcaption>
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<b>Fig. 1</b>: Biofilms composed by CsgASpyCatcherHisTag subunits. After scrutinization, biofilm can viewed around the '''E.coli'''
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<b>Fig. 1</b>: Biofilms composed by His-CsgA-SpyCacher-histag subunits. After scrutinization, biofilm can viewed around the '''E.coli'''
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
  
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<h4> Fluoresence Binding Test </h4>
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<h4>QDs’ Fluoresence Binding Test </h4>
 
<p>
 
<p>
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In order to test the effect of binding between CsgASpyCatcherHisTag mutant and inorganic nanoparticles, we apply same amount of suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 30-min incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, CsgASpyCatcherHisTag mutant were induced and thus secreted biofilm, and firmly attached with QDS and thus show bright fluorescence. Therefore, we ensure the stable coordinate bonds between CsgASpyCatcherHisTag mutant and QDs can manage to prevent QDs from being taken away by liquid flow.  
+
In order to test the effect of binding between His-CsgA-SpyCacher-histag mutant and inorganic nanoparticles, we apply same amount of suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 30-min incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, His-CsgA-SpyCacher-histag mutant was induced and secreted biofilm, and firmly attached with QDS and thus showed bright fluorescence. Therefore, we ensure the stable coordinate bonds between His-CsgA-SpyCacher-histag mutant and QDs can manage to prevent QDs from being taken away by liquid flow.  
 
</p>
 
</p>
  
 
<figure align="center">
 
<figure align="center">
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<img src="https://static.igem.org/mediawiki/parts/5/5f/Shanghaitechchina_SCH%2BQD.tiff" width="70%" height="70%">
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<img src="https://static.igem.org/mediawiki/parts/5/56/Shanghaitechchina_hisCsgASpyCatcherHistag%2BQD.png" width="50%">
 
<figcaption>
 
<figcaption>
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<b>Fig. 2</b>: Binding between CsgASpyCatcherHisTag mutant and inorganic QDs.
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<b>Fig. 2</b>: Binding between His-CsgA-SpyCacher-histag mutant and inorganic CdS QDs.
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
  
  
−
<h4> TEM: visualization of binding test</h4>
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<h4> TEM: visualization of binding test with AuNPs</h4>
 
<p>
 
<p>
−
transmission electron microscopy(TEM) visualize the binding effect of CsgASpyCatcherHisTag mutant E.coli with AuNPs. From the picture, it shows biofilm areas are attached by AuNPs and thus confirm the viability of histag on CsgASpyCatcherHisTag mutant biofilm.
+
transmission electron microscopy(TEM) visualize the binding effect of His-CsgA-SpyCacher-histag mutant E.coli with AuNPs. From the picture, it shows biofilm areas are attached by AuNPs and thus confirm the viability of histag on His-CsgA-SpyCacher-histag mutant biofilm.
 
</p>
 
</p>
  
 
<figure align="center">
 
<figure align="center">
−
<img src="https://static.igem.org/mediawiki/parts/0/08/Shanghaitechchina_CsgASpyCatcherHistag%2BAu.png" width="50%" height="50%" >
+
<img src="https://static.igem.org/mediawiki/parts/e/e8/Shanghaitechchina_HSCH%2BAu.png" width="40%" >
 +
 
 +
 
 +
<figcaption>
 +
<b>Fig. 3</b>: Biofilms composed by His-CsgA-SpyCacher-histag subunits bind with AuNPs.
 +
</figcaption>
 +
</figure>
 +
<h4>Spy System Functional Test</h4>
 +
<p>As figure illustrated, his-CsgA-SpyCatcher-his mutant incubated with mcherry-SpyTag show a clear biofilm-associated mcherry fluorescence signal, which indicates the accurate conformation and function of the SpyTag and SpyCatcher linkage system. The third figure is merged by the first and second figures of each sample are snapped respectively under green laser field with 558 nm wavelength and bright field of fluorescence microscopy, Zeiss Axio Imager Z2. As for controls, strains secreted CsgA–Histag and ΔCsgA both are unable to specifically attach to SpyTag thus no distinct localization highlight of red fluorescence on E.coli. That to a large extent proved the specificity of our desired linkage between SpyTag and SpyCatcher system. </p>
 +
<figure align="center">
 +
<img src="https://static.igem.org/mediawiki/parts/9/9f/Shanghaitechchina_spy_part.png" width="60%" height="60%" >
 
<figcaption>
 
<figcaption>
−
<b>Fig. 3</b>: Biofilms composed by CsgASpyCatcherHisTag subunits bind with AuNPs.
+
<b>Fig. 3</b>: The first figures of each sample are snapped under green laser of 558 nm wavelength and mcherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
  
 
</html>
 
</html>

Latest revision as of 11:55, 19 October 2016

CsgASpyCatcherHisTag

This is the subunit of major proteins of E. Coli biofilm Curli system linked to SpyCatcher and HisTag. The two added protein tag and domain can bring new functions to biofilms . For example, SpyTag can covalently interact with any fusion protein containing Spytcatcher, While His tag can facilitate purification, and endow biofilms with specific binding ability towards NTA-decorated nanoparticles, as demonstrated below

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 841
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Four different experiments were conducted to characterize this biobrick:

  • TEM: visualization of His-CsgA-SpyCacher-histag mutant biofilm
  • QDs’ Fluorescence Binding Test
  • TEM: visualization of binding test with AuNPs
  • Spy System Functional Test

TEM: visualization of His-CsgA-SpyCacher-histag mutant biofilm

Fig. 1: Biofilms composed by His-CsgA-SpyCacher-histag subunits. After scrutinization, biofilm can viewed around the '''E.coli'''

QDs’ Fluoresence Binding Test

In order to test the effect of binding between His-CsgA-SpyCacher-histag mutant and inorganic nanoparticles, we apply same amount of suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 30-min incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, His-CsgA-SpyCacher-histag mutant was induced and secreted biofilm, and firmly attached with QDS and thus showed bright fluorescence. Therefore, we ensure the stable coordinate bonds between His-CsgA-SpyCacher-histag mutant and QDs can manage to prevent QDs from being taken away by liquid flow.

Fig. 2: Binding between His-CsgA-SpyCacher-histag mutant and inorganic CdS QDs.

TEM: visualization of binding test with AuNPs

transmission electron microscopy(TEM) visualize the binding effect of His-CsgA-SpyCacher-histag mutant E.coli with AuNPs. From the picture, it shows biofilm areas are attached by AuNPs and thus confirm the viability of histag on His-CsgA-SpyCacher-histag mutant biofilm.

Fig. 3: Biofilms composed by His-CsgA-SpyCacher-histag subunits bind with AuNPs.

Spy System Functional Test

As figure illustrated, his-CsgA-SpyCatcher-his mutant incubated with mcherry-SpyTag show a clear biofilm-associated mcherry fluorescence signal, which indicates the accurate conformation and function of the SpyTag and SpyCatcher linkage system. The third figure is merged by the first and second figures of each sample are snapped respectively under green laser field with 558 nm wavelength and bright field of fluorescence microscopy, Zeiss Axio Imager Z2. As for controls, strains secreted CsgA–Histag and ΔCsgA both are unable to specifically attach to SpyTag thus no distinct localization highlight of red fluorescence on E.coli. That to a large extent proved the specificity of our desired linkage between SpyTag and SpyCatcher system.

Fig. 3: The first figures of each sample are snapped under green laser of 558 nm wavelength and mcherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.