Difference between revisions of "Part:BBa K2132001"

 
 
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__NOTOC__
 
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<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
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2132001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2132001 SequenceAndFeatures</partinfo>
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<html>
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<h3>Characterization</h3>
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<p>
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Four different experiments were conducted to characterize this biobrick:
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<ul>
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<li> TEM: visualization of His-CsgA-SpyCacher-histag mutant biofilm </li>
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<li> QDs’ Fluorescence Binding Test </li>
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<li> TEM: visualization of binding test with AuNPs </li>
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<li> Spy System Functional Test </li></ul>
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</p>
  
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<h4> TEM: visualization of His-CsgA-SpyCacher-histag mutant biofilm </h4>
  
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<figure align="center">
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===Functional Parameters===
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<img src="https://static.igem.org/mediawiki/parts/7/7d/Shanghaitechchina_hsch_part.png" width="50%" height="50%">
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<partinfo>BBa_K2132001 parameters</partinfo>
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<figcaption>
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<!-- -->
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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'''
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</figcaption>
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</figure>
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<h4>QDs’ Fluoresence Binding Test </h4>
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<p>
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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.
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</p>
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<figure align="center">
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<img src="https://static.igem.org/mediawiki/parts/5/56/Shanghaitechchina_hisCsgASpyCatcherHistag%2BQD.png" width="50%">
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<figcaption>
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<b>Fig. 2</b>: Binding between His-CsgA-SpyCacher-histag mutant and inorganic CdS QDs.
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</figcaption>
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</figure>
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<h4> TEM: visualization of binding test with AuNPs</h4>
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<p>
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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.
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</p>
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<figure align="center">
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<img src="https://static.igem.org/mediawiki/parts/e/e8/Shanghaitechchina_HSCH%2BAu.png" width="40%" >
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<figcaption>
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<b>Fig. 3</b>: Biofilms composed by His-CsgA-SpyCacher-histag subunits bind with AuNPs.
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</figcaption>
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</figure>
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<h4>Spy System Functional Test</h4>
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<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>
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<figure align="center">
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<img src="https://static.igem.org/mediawiki/parts/9/9f/Shanghaitechchina_spy_part.png" width="60%" height="60%" >
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<figcaption>
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<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.
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</figcaption>
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</figure>
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</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.