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<img src=" https://static.igem.org/mediawiki/parts/6/68/Shanghaitechchina_dye.png " width="55%"> | <img src=" https://static.igem.org/mediawiki/parts/6/68/Shanghaitechchina_dye.png " width="55%"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 1. </b> Crystal violet and Congo Red reagent.</p> |
<h4><b>TEM</b></h4> | <h4><b>TEM</b></h4> | ||
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<img src=" https://static.igem.org/mediawiki/parts/2/26/Shanghaitechchina_TEM_device.png" width="55%"> | <img src=" https://static.igem.org/mediawiki/parts/2/26/Shanghaitechchina_TEM_device.png" width="55%"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 2. </b> TEM device at the National Center for Protein Science Shanghai.</p> |
<h4><b>Quantum Dots Binding Assay</b></h4> | <h4><b>Quantum Dots Binding Assay</b></h4> | ||
Mechanisms of Quantum dots binding assay have been introduced in detail in Quantum Dots part. we utilizing Co/Ni-NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) to bind with the histidine in Histags on our biofilm and thus characterize its formation. The whole linkage is performed by forming firm coordinate bonds. They could be applied to quick detection of biofilm expression of His-tagged proteins with naked eye under UV light owing to the photoluminescence of QDs, and accurate concentration measurement under fluorescence spectrum (A detailed protocol for repeatable measurements is included in our Wikipage). <p></p> | Mechanisms of Quantum dots binding assay have been introduced in detail in Quantum Dots part. we utilizing Co/Ni-NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) to bind with the histidine in Histags on our biofilm and thus characterize its formation. The whole linkage is performed by forming firm coordinate bonds. They could be applied to quick detection of biofilm expression of His-tagged proteins with naked eye under UV light owing to the photoluminescence of QDs, and accurate concentration measurement under fluorescence spectrum (A detailed protocol for repeatable measurements is included in our Wikipage). <p></p> | ||
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<img src="https://static.igem.org/mediawiki/parts/9/95/Shanghaitechchina_CsgAhis_CR.png" style="width:80%;"> | <img src="https://static.igem.org/mediawiki/parts/9/95/Shanghaitechchina_CsgAhis_CR.png" style="width:80%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 3.</b>Congo red assay of CsgA-Histag on YESCA plates</p> |
The figures shown above point out that the CsgA-Histag mutant induced by 0.25 μg/ ml of aTc will produce amyloid structures which are dyed to red by CR in comparison to the negative control. This assay indicates the success in expression of the self-assembly to curli fibers. <p></p> | The figures shown above point out that the CsgA-Histag mutant induced by 0.25 μg/ ml of aTc will produce amyloid structures which are dyed to red by CR in comparison to the negative control. This assay indicates the success in expression of the self-assembly to curli fibers. <p></p> | ||
<h4><b>2. Crystal Violet Assay: quantification test of biofilm </b></h4> | <h4><b>2. Crystal Violet Assay: quantification test of biofilm </b></h4> | ||
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<img src="https://static.igem.org/mediawiki/parts/b/bc/Shanghaitechchina_crystalviolethistag.png" style="width:60%;"> | <img src="https://static.igem.org/mediawiki/parts/b/bc/Shanghaitechchina_crystalviolethistag.png" style="width:60%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 4.</b>:Crystal violet assay of CsgA-Histag.</p> |
<h4><b>3. Quantum dots fluorescence test: binding test of Histag with nanomaterials</b></h4> | <h4><b>3. Quantum dots fluorescence test: binding test of Histag with nanomaterials</b></h4> | ||
<b>new characterization of the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1583003">PART BBa_K1583003</a></b><p></p> | <b>new characterization of the <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1583003">PART BBa_K1583003</a></b><p></p> | ||
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<img src="https://static.igem.org/mediawiki/parts/f/f2/Shanghaitechchina_Histag%2BQDs.png" style="width:60%;"> | <img src="https://static.igem.org/mediawiki/parts/f/f2/Shanghaitechchina_Histag%2BQDs.png" style="width:60%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 5.</b> Fluorescence test of CsgA-His binding with nanomaterials</p> |
<h4><b>4. TEM: visualization of binding test</b></h4> | <h4><b>4. TEM: visualization of binding test</b></h4> | ||
Since biofilm nanofibers are thin and inconspicuous against the background, we harness CdSe QDs binding to highlight the biofilm area. The first image illustrates biofilm areas which are densely covered by QDs after induced for 72h and incubated, compared to the second image which is not incubated with nanoparticles CdSe. The third one is a negative control without inducer, bacteria scattered without forming biofilm<p></p> | Since biofilm nanofibers are thin and inconspicuous against the background, we harness CdSe QDs binding to highlight the biofilm area. The first image illustrates biofilm areas which are densely covered by QDs after induced for 72h and incubated, compared to the second image which is not incubated with nanoparticles CdSe. The third one is a negative control without inducer, bacteria scattered without forming biofilm<p></p> | ||
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<img src="https://static.igem.org/mediawiki/parts/f/f0/Shanghaitechchina_CsgAHistag%2BQD.png" style="width:100%;"> | <img src="https://static.igem.org/mediawiki/parts/f/f0/Shanghaitechchina_CsgAHistag%2BQD.png" style="width:100%;"> | ||
<p style="text-align:center"> | <p style="text-align:center"> | ||
− | <b> | + | <b>Fig 6.</b>:Representative TEM images of biotemplated CdSe quantum dots on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with QDs for 1h, nanomaterials are densely attached to biofilm. |
</p> | </p> | ||
Finally, transmission electron microscopy(TEM) visualize the binding effect of CsgA-Histag mutant E.coli with CdS nanorods in comparison with image of pure nanofiber composed by CsgA-Histag and one without inducer. As can be clearly seen from the figures, with inducer there’s distinct nanofibers outside the bacteria contrast to the third picture in which E.coli are not induced. From the first picture, it shows biofilm areas organize CdS nanorods around the bacteria and we confirm the viability of bio-abiotic hybrid system.<p></p> | Finally, transmission electron microscopy(TEM) visualize the binding effect of CsgA-Histag mutant E.coli with CdS nanorods in comparison with image of pure nanofiber composed by CsgA-Histag and one without inducer. As can be clearly seen from the figures, with inducer there’s distinct nanofibers outside the bacteria contrast to the third picture in which E.coli are not induced. From the first picture, it shows biofilm areas organize CdS nanorods around the bacteria and we confirm the viability of bio-abiotic hybrid system.<p></p> | ||
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<img src="https://static.igem.org/mediawiki/parts/e/e1/Shanghaitechchina_CsgAHistag%2Bnanorods.png" style="width:100%;"> | <img src="https://static.igem.org/mediawiki/parts/e/e1/Shanghaitechchina_CsgAHistag%2Bnanorods.png" style="width:100%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 7.</b>Representative TEM images of biotemplated CdS nanorods on CsgA-His. </p> |
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<img src="https://static.igem.org/mediawiki/parts/0/05/Shanghaitechchina_HISCsgASpyCatcher_CR.png" style="width:60%;align:center"> | <img src="https://static.igem.org/mediawiki/parts/0/05/Shanghaitechchina_HISCsgASpyCatcher_CR.png" style="width:60%;align:center"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 8.</b> Congo Red Assay of His-CsgA-SpyCatcher-Histag</p> |
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<img src="https://static.igem.org/mediawiki/parts/c/c8/Shanghaitechchina_hisCsgASpyCatcherHistag_CR.png" style="width:100%;"> | <img src="https://static.igem.org/mediawiki/parts/c/c8/Shanghaitechchina_hisCsgASpyCatcherHistag_CR.png" style="width:100%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 9.</b> Congo Red Assay of His-CsgA-SpyCatcher</p> |
</div> | </div> | ||
<div class="col-lg12"> | <div class="col-lg12"> | ||
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<img src="https://static.igem.org/mediawiki/parts/5/56/Shanghaitechchina_hisCsgASpyCatcherHistag%2BQD.png" style="width:60%;"> | <img src="https://static.igem.org/mediawiki/parts/5/56/Shanghaitechchina_hisCsgASpyCatcherHistag%2BQD.png" style="width:60%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 10.</b> Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.</p> |
<b>His-CsgA-SpyCatcher</b><p></p> | <b>His-CsgA-SpyCatcher</b><p></p> | ||
Using the same approach, we also conducted binding assay of His-CsgA-SpyCatcher with QDs to characterize the expression of biofilm and the visual result shows vividly that His-CsgA-SpyCatcher can bind successfully with the QDs with the existence of inducer aTc, which shows the functional similarity in CsgA-Histag. The picture was snapped by BioRad ChemiDoc MP, false colored.<p></p> | Using the same approach, we also conducted binding assay of His-CsgA-SpyCatcher with QDs to characterize the expression of biofilm and the visual result shows vividly that His-CsgA-SpyCatcher can bind successfully with the QDs with the existence of inducer aTc, which shows the functional similarity in CsgA-Histag. The picture was snapped by BioRad ChemiDoc MP, false colored.<p></p> | ||
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<img src="https://static.igem.org/mediawiki/parts/4/45/Shanghaitechchina_hisCsgASpyCatcher%2BQD.png" style="width:60%;"> | <img src="https://static.igem.org/mediawiki/parts/4/45/Shanghaitechchina_hisCsgASpyCatcher%2BQD.png" style="width:60%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 11.</b> Quantum dots templating assay on His-CsgA-SpyCatcher biofilm.</p> |
</div> | </div> | ||
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<img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" style="width:100%;"> | <img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" style="width:100%;"> | ||
</center> | </center> | ||
− | <p style="text-align:center"><b> | + | <p style="text-align:center"><b>Fig 12. </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.</p> |
</div> | </div> | ||
</div> | </div> | ||
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</center> | </center> | ||
<p style="text-align:center"> | <p style="text-align:center"> | ||
− | <b> | + | <b>Fig 13. </b>Nanomaterial binding test. Images were shot by iPhone 5s under 365nm UV light, Tanon UV-100 |
</p> | </p> | ||
We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg/ ml of aTc will induce the best expression performance of biofilm. The possible reason for higher concentration of inducer strangely leading into low production of biofilm might lie in that aTc, a kind of antibiotic, can be harmful to protein synthesis in bacteria. We speculate there’s an antagonism between the effect of promoting expression and impeding growth brought by aTc and 0.25 μg/ ml of aTc just reach the optimal point.<p></p> | We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg/ ml of aTc will induce the best expression performance of biofilm. The possible reason for higher concentration of inducer strangely leading into low production of biofilm might lie in that aTc, a kind of antibiotic, can be harmful to protein synthesis in bacteria. We speculate there’s an antagonism between the effect of promoting expression and impeding growth brought by aTc and 0.25 μg/ ml of aTc just reach the optimal point.<p></p> |
Revision as of 15:12, 18 October 2016