Difference between revisions of "Part:BBa K2132001"
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<li> TEM: visualization of CsgASpyCatcherHisTag mutant biofilm </li>
 
<li> TEM: visualization of CsgASpyCatcherHisTag mutant biofilm </li>
 
<li> Fluoresence 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 </li>
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<li> Spy System Functional Test </li></ul>
 
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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%">
 
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<b>Fig. 1</b>: Biofilms composed by CsgASpyCatcherHisTag subunits. After scrutinization, biofilm can viewed around the '''E.coli'''
 
<b>Fig. 1</b>: Biofilms composed by CsgASpyCatcherHisTag subunits. After scrutinization, biofilm can viewed around the '''E.coli'''
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<b>Fig. 3</b>: Biofilms composed by CsgASpyCatcherHisTag subunits bind with AuNPs.
 
<b>Fig. 3</b>: Biofilms composed by CsgASpyCatcherHisTag subunits bind with AuNPs.
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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 indicating 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 prove the specificity of our desired linkage between SpyTag and SpyCatcher system. </p>
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<img src="https://static.igem.org/mediawiki/parts/9/9f/Shanghaitechchina_spy_part.png" width="100%" height="100%" >
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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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Revision as of 10:35, 16 October 2016


CsgASpyCatcherHisTag

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.

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

Three different experiments were conducted to characterize these biobricks:

  • TEM: visualization of CsgASpyCatcherHisTag mutant biofilm
  • Fluoresence Binding Test
  • TEM: visualization of binding test
  • Spy System Functional Test

TEM: visualization of CsgASpyCatcherHisTag mutant biofilm

Fig. 1: Biofilms composed by CsgASpyCatcherHisTag subunits. After scrutinization, biofilm can viewed around the '''E.coli'''

Fluoresence Binding Test

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.

Fig. 2: Binding between CsgASpyCatcherHisTag mutant and inorganic QDs.

TEM: visualization of binding test

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.

Fig. 3: Biofilms composed by CsgASpyCatcherHisTag 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 indicating 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 prove 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.