Difference between revisions of "Team:ShanghaitechChina/Proof"

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  1. Successful production and characterization of engineered Biofilms, demonstrating that engineered biofilms composed of CsgA-Histag  fused protein allowed firm binding of semiconductor nanomaterials.  <b><a href="http://parts.igem.org/Part:BBa_K2132001">BioBrick BBa_K2132001</a></b> <p></p>
 
  1. Successful production and characterization of engineered Biofilms, demonstrating that engineered biofilms composed of CsgA-Histag  fused protein allowed firm binding of semiconductor nanomaterials.  <b><a href="http://parts.igem.org/Part:BBa_K2132001">BioBrick BBa_K2132001</a></b> <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 the 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>   
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  2.Next question is how to ensure normal enzyme activity by making these four enzymes can express simultaneously under a moderate level? So the 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, we need our enzyme to be functional to make a precondition for our whole plan. By the way, we can test the advantage of Acembl system vs pETDuet-1+pCDFDuet-1 system.So we observed successful hydrogen production with freely-flowing CdS Nanorods and find a higher enzyme catalysis activity. <p></p>
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  3.Finally, is our enzyme to be functional to make a precondition for our whole plan? By the way, is there any advantage of our device base on Acembl system vs pETDuet-1+pCDFDuet-1 system in previous study? So we observed successful hydrogen production with freely-flowing CdS Nanorods and find a higher enzyme catalysis activity. <p></p>
  
 
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<div id="CHydrogenase"class="col-lg-12">
 
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<h3>Hydrogenese gene clusters</h3><p></p>
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<h4><b>Hydrogenese gene clusters</b></h4><p></p>
 
  <p>High-activity hydrogenase is necessary for our system. To achieve efficient enzymatic activities, we codon-optimized and constructed the whole hydrogenase gene clusters (from Clostridium Acetobutylicum) by leveraging the multi-expression Acembl System.  Please refer to <b><a href="https://2016.igem.org/Team:ShanghaitechChina/Hydrogen">Hydrogenase Session</a></b> for more details. </p>   
 
  <p>High-activity hydrogenase is necessary for our system. To achieve efficient enzymatic activities, we codon-optimized and constructed the whole hydrogenase gene clusters (from Clostridium Acetobutylicum) by leveraging the multi-expression Acembl System.  Please refer to <b><a href="https://2016.igem.org/Team:ShanghaitechChina/Hydrogen">Hydrogenase Session</a></b> for more details. </p>   
 
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<p style="text-align:center"><b>Figure 4E</b> Fusion of the plasmid in step (4C) and plasmid 3.</p>
 
<p style="text-align:center"><b>Figure 4E</b> Fusion of the plasmid in step (4C) and plasmid 3.</p>
 
For a whole fused plasmid, It becomes hard to analyze it with just Xho I single enzyme. The bar at 3k actually accounts for two bars, with a separation of 20bp. In the picture, although the four bands predicted by SnapGene® can be found on our real gel, it is less clear. <p></p>
 
For a whole fused plasmid, It becomes hard to analyze it with just Xho I single enzyme. The bar at 3k actually accounts for two bars, with a separation of 20bp. In the picture, although the four bands predicted by SnapGene® can be found on our real gel, it is less clear. <p></p>
Given the inconvenience with testing by restriction, we turned to resistance screening. The result is that it is resistant to four antibodies (Ampicillin, Chloramphenicol, kanamycin and Spectinomycin). Figure 4E shows that plasmids obtained in step 2 and plasmid 4 are successfully fused. Thus, we obtained a plasmid with all four subunits, HydA, HydE, HydF, HydG, fused together. The next step is to induce the expression of the hydrogenase.<p></p>
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Given the inconvenience with testing by restriction, we turned to resistance screening. The result is that it is resistant to four antibodies (Ampicillin, Chloramphenicol, kanamycin and Spectinomycin).  
  
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<h4><b>Expression of the hydrogenase.</b></h4><p></p>
  
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As we had successfully get the device,the next step is to induce the expression of the hydrogenase.<p></p>
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<center><img src="https://static.igem.org/mediawiki/2016/8/8e/T--ShanghaitechChina--hrduogenase--paojiao.jpg"></center>
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To see, we use the antibody of Histag to show the specific of HydA-spycatcher and HydA-spytag and got the result. While we can not avoid the other protein with a similar affinity.
  
 
<p id="AResults"></p>
 
<p id="AResults"></p>

Revision as of 15:08, 19 October 2016

igem2016:ShanghaiTech