Difference between revisions of "Part:BBa K2132001"
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<h4>QDs’ Fluoresence Binding Test </h4> | <h4>QDs’ Fluoresence Binding Test </h4> | ||
<p> | <p> | ||
| − | 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 | + | 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> | ||
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</figure> | </figure> | ||
<h4>Spy System Functional Test</h4> | <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 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 | + | <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 proved the specificity of our desired linkage between SpyTag and SpyCatcher system. </p> |
<figure align="center"> | <figure align="center"> | ||
<img src="https://static.igem.org/mediawiki/parts/9/9f/Shanghaitechchina_spy_part.png" width="60%" height="60%" > | <img src="https://static.igem.org/mediawiki/parts/9/9f/Shanghaitechchina_spy_part.png" width="60%" height="60%" > | ||
Revision as of 15:42, 17 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 841
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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
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.
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.
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 proved the specificity of our desired linkage between SpyTag and SpyCatcher system.
