Difference between revisions of "Team:ShanghaitechChina/Proof"
m |
LechenQian (Talk | contribs) |
||
| Line 60: | Line 60: | ||
<h1 align="center">Major Achievements in Constructing Our Device</h1> | <h1 align="center">Major Achievements in Constructing Our Device</h1> | ||
| − | 1. Successful production and characterization of engineered Biofilms, demonstrating that engineered biofilms composed of CsgA | + | 1. Successful production and characterization of engineered Biofilms, demonstrating that engineered biofilms composed of CsgA fused protein allowed firm binding of semiconductor nanomaterials. <p></p> |
| − | 2.Next question is how to ensure normal enzyme activity by making these four enzymes can express simultaneously under a moderate level? So | + | Base on this conception, we constructed two major device in our project and tested their function:<p></p> |
| − | 3.Finally, is our enzyme to be functional to make a precondition for our whole plan? | + | <ul> |
| + | <li>CsgA-Histag</li> | ||
| + | <li>His-CsgA-SpyCatcher-Histag: <a href="http://parts.igem.org/Part:BBa_K2132001">BioBrick BBa_K2132001</a></li> | ||
| + | </ul> | ||
| + | Please refer to <a href="https://2016.igem.org/Team:ShanghaitechChina/Biofilm"><b>Engineered Biofilms</b></a> for details of the successful construction and characterization of engineered biofilms | ||
| + | <p></p> | ||
| + | 2.Next question is how to ensure normal enzyme activity by making these four enzymes can express simultaneously under a moderate level? So we built the device based on Acembl system and we make successful integration of [FeFe]-hydrogenase gene clusters from Clostridium acetobutylicum into one single plasmid to allow reliable expression. <p></p> Please refer to <b><a href="https://2016.igem.org/Team:ShanghaitechChina/Hydrogen">Hydrogenase Session</a></b> for more details. </p> | ||
| + | 3.Finally, is our enzyme to be functional to make a precondition for our whole plan? Here we can show you successful hydrogen production with freely-flowing CdS Nanorods <p></p> | ||
</div> | </div> | ||
| Line 72: | Line 79: | ||
<div class="row"> | <div class="row"> | ||
<div class="col-lg-12"> | <div class="col-lg-12"> | ||
| − | <h1 align="center">Engineered Biofilm Device</h1> | + | <h1 align="center">Engineered Biofilm Device 1</h1> |
<h3>A. To allow easy recycling of precious semiconductor nanomaterials, we utilized engineered biofilms to anchor nanomaterials via metal coordination chemistry. Please refer to <a href="https://2016.igem.org/Team:ShanghaitechChina/Biofilm"><b>Engineered Biofilms</b></a> for details of the successful construction and characterization of engineered biofilms that allow firm binding of nanomaterials. Key data are reproduced below. </h3> <p></p> | <h3>A. To allow easy recycling of precious semiconductor nanomaterials, we utilized engineered biofilms to anchor nanomaterials via metal coordination chemistry. Please refer to <a href="https://2016.igem.org/Team:ShanghaitechChina/Biofilm"><b>Engineered Biofilms</b></a> for details of the successful construction and characterization of engineered biofilms that allow firm binding of nanomaterials. Key data are reproduced below. </h3> <p></p> | ||
| − | + | <h3><strong>Device 1</strong></h3> | |
| − | <h4> First, | + | <h4> <strong>First, we constructed and test CsgA-Histag expression and binding validity with nanomaterials under inducer aTc. </strong></h4> |
<h4 ><b>1. Congo Red：successful secretion and expression</b></h4> | <h4 ><b>1. Congo Red：successful secretion and expression</b></h4> | ||
<p></p> | <p></p> | ||
<center> | <center> | ||
| − | <img src="https://static.igem.org/mediawiki/parts/9/95/Shanghaitechchina_CsgAhis_CR.png" style="width: | + | <img src="https://static.igem.org/mediawiki/parts/9/95/Shanghaitechchina_CsgAhis_CR.png" style="width:45%;"> |
</center> | </center> | ||
<p style="text-align:center"><b>Fig 1.</b> Fluorescence test of CsgA-His binding with nanomaterials</p> | <p style="text-align:center"><b>Fig 1.</b> Fluorescence test of CsgA-His binding with nanomaterials</p> | ||
| − | This assay | + | This assay indicated the success in expression of the self-assembly 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> | ||
| − | Further, we | + | Further, we used crystal violet assay to obtain quantitative data about the relative density of cells and biofilm adhesion to multi-wells cluster dishes. |
<p></p> | <p></p> | ||
<center> | <center> | ||
| Line 95: | Line 102: | ||
</center> | </center> | ||
<p style="text-align:center"><b>Fig 2.</b>Crystal violet assay of CsgA-Histag.</p> | <p style="text-align:center"><b>Fig 2.</b>Crystal violet assay of CsgA-Histag.</p> | ||
| − | This assay indicates the success in expression of the self-assembly curli fibers. | + | This assay indicates the success in expression of the self-assembly curli fibers. The difference between induced strains secreted CsgA-Histag and ΔCsgA manifest a distinct extracellular biofilm production in the modified strain. <p></p> |
| − | + | ||
| − | + | <h4 ><b>3.Quantum dots fluorescence test: successful binding test of Histag with nanomaterials (CdSeS/CdSe/ZnS core/shell quantum dots) in macroscopoc world</b></h4> | |
| − | <h4 ><b>3.Quantum dots fluorescence test: successful binding test of Histag | + | |
<p></p> | <p></p> | ||
After confirming that our parts success in biofilm expression, we are going to test the effect of binding between CsgA-Histag mutant and inorganic nanoparticles. The result was consistent with our anticipation: On the left, CsgA-Histag mutant were induced and QDs are attached with biofilms, thus show bright fluorescence. Therefore, we ensure the stable coordinate bonds between CsgA-Histag mutant and QDs.The picture was snapped by ChemiDoc MP,BioRad, false colored.<p></p> | After confirming that our parts success in biofilm expression, we are going to test the effect of binding between CsgA-Histag mutant and inorganic nanoparticles. The result was consistent with our anticipation: On the left, CsgA-Histag mutant were induced and QDs are attached with biofilms, thus show bright fluorescence. Therefore, we ensure the stable coordinate bonds between CsgA-Histag mutant and QDs.The picture was snapped by ChemiDoc MP,BioRad, false colored.<p></p> | ||
| Line 110: | Line 115: | ||
<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. <p></p> | Since biofilm nanofibers are thin and inconspicuous against the background, we harness CdSe QDs binding to highlight the biofilm area. <p></p> | ||
| − | < | + | <center> |
<img src="https://static.igem.org/mediawiki/parts/f/f0/Shanghaitechchina_CsgAHistag%2BQD.png" style="width:80%;"> | <img src="https://static.igem.org/mediawiki/parts/f/f0/Shanghaitechchina_CsgAHistag%2BQD.png" style="width:80%;"> | ||
| + | </center> | ||
<p style="text-align:center"> | <p style="text-align:center"> | ||
<b>Fig 4.</b>Representative TEM images of biotemplated CdS quantum dots on CsgA-His. | <b>Fig 4.</b>Representative TEM images of biotemplated CdS quantum dots on CsgA-His. | ||
| Line 127: | Line 133: | ||
<div class="row"> | <div class="row"> | ||
<div class="col-lg-12"> | <div class="col-lg-12"> | ||
| − | <h1 align="center">Engineered Biofilm Device</h1> | + | <h1 align="center">Engineered Biofilm Device 2</h1> |
| − | + | <h3><strong>Device 2</strong></h3> | |
| − | <h4> Second, | + | <h4> Second, we constructed His-CsgA-SpyCatcher-Histag device and successfully characterized its expression and binding effect through the following five steps. we want to achieve the complex design with extra function of binding SpyTag-linked enzymes in addition to its nanomaterial-binding through Histag. This is realized with our Part BBa_K2132001 under the promoter of aTc. </h4> |
<h4><b>1. Congo Red：successful secretion and expression</b></h4> | <h4><b>1. Congo Red：successful secretion and expression</b></h4> | ||
After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg ml-1 of aTc and cultured for 72h at 30℃ successfully secreted a thin-layer biofilm on the plate which are stained to brown-red color by CR. This assay also proved that the new and challenging construction of appending a large protein onto CsgA subunits will work accurately and effectively.<p></p> | After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg ml-1 of aTc and cultured for 72h at 30℃ successfully secreted a thin-layer biofilm on the plate which are stained to brown-red color by CR. This assay also proved that the new and challenging construction of appending a large protein onto CsgA subunits will work accurately and effectively.<p></p> | ||
| − | <div class="col-lg- | + | <div class="col-lg-12"> |
<center> | <center> | ||
| − | <img src="https://static.igem.org/mediawiki/parts/0/05/Shanghaitechchina_HISCsgASpyCatcher_CR.png" style="width: | + | <img src="https://static.igem.org/mediawiki/parts/0/05/Shanghaitechchina_HISCsgASpyCatcher_CR.png" style="width:45%;"> |
</center> | </center> | ||
<p style="text-align:center"><b>Fig 5.</b> Congo Red Assay of His-CsgA-SpyCatcher-Histag.</p> | <p style="text-align:center"><b>Fig 5.</b> Congo Red Assay of His-CsgA-SpyCatcher-Histag.</p> | ||
| Line 142: | Line 148: | ||
<div class="col-lg-12"> | <div class="col-lg-12"> | ||
| − | <h4><b>2. Quantum dots fluorescence test: successful binding test of Histag | + | <h4><b>2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials</b></h4> |
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. We use His-CsgA-SpyCatcher-Histag as demo. <p></p> | 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. We use His-CsgA-SpyCatcher-Histag as demo. <p></p> | ||
</div> | </div> | ||
| Line 180: | Line 186: | ||
<p style="text-align:center"><b>Fig 9.</b>mcherry-SpyTag fluorescence protein binding test of His-CsgA-SpyCatcher-(Histag). </p> | <p style="text-align:center"><b>Fig 9.</b>mcherry-SpyTag fluorescence protein binding test of His-CsgA-SpyCatcher-(Histag). </p> | ||
</div> | </div> | ||
| − | < | + | |
| + | <h3>So far above, we proved that two engineered biofilm devices function properly. Later, we tested different inducer concentration gradient to find out the best induction condition.</h3> | ||
| + | <h3><b>Inducer concentration optimization</b></h3> | ||
We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg ml-1 of aTc will induce the best expression performance of biofilm, which is exactly the inducer concentration we applied in the project. | We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg ml-1 of aTc will induce the best expression performance of biofilm, which is exactly the inducer concentration we applied in the project. | ||
<p></p> | <p></p> | ||
Revision as of 17:38, 19 October 2016
