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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1. Congo Red：successful secretion and expression
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<h4> TEM: visualization of His-CsgA-SpyCacher-histag mutant biofilm </h4>
  
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After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg ml-1 of aTc successfully secreted a thin-layer biofilm on the plate which are stained to brown-red color by CR, compared to the negative control with no inducer. (Because the ratio between Congo Red dye and Brilliant Blue dye is not in the best state which leads to the unapparent phenomenon through the lens, the brown red biofilm is easy to be identified visually.) This assay also proved that the new and challenging construction of appending a large protein onto CsgA subunits will work accurately and effectively.
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<figure align="center">
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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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<figcaption>
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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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2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials
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<h4>QDs’ Fluoresence Binding Test </h4>
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Then comes to the next part: we want to check if SpyCatcher protein will be too large to cause steric hindrance effect on Histag peptide. The best approach to verify is the fluorescence assay of binding with nanomaterials.  
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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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After applying the same steps as introduced above, the bottom of left well show a large area of bright fluorescence, manifesting His-CsgA-SpyCatcher-Histag mutant secreted biofilms under the control of inducer and Histag on it is not blocked, what is more, it is firmly attached with QDs. From this assay, we assure that the SpyCatcher will not impose negative effect on the binding between inorganic material and biofilm. The picture was snapped by ChemiDoc MP, BioRad.
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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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3. TEM: visualization of binding test
 
  
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===Functional Parameters===
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<h4> TEM: visualization of binding test with AuNPs</h4>
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<partinfo>BBa_K2132001 parameters</partinfo>
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<p>
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<!-- -->
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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.