Difference between revisions of "Part:BBa K2132004"

 
 
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<partinfo>BBa_K2132004 short</partinfo>
 
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This is a modified protein, we added Histag and TEV site to the N-terminal site of hydA, and SpyCatcher to the C-terminal site of hydA.
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This part encodes the major enzyme of [FeFe] hydrogenase gene clusters (refer to HydA), originally from the bacterium Clostridium acetobutylicum (Original coding sequence: hydA, BBa_K535002, Designed by: iGEM11_UNAM-Genomics_ Mexico.)  In particular, this codon-optimized protein was purposely added with Histag and TEV site to the N-terminal site of HydA, and SpyCatcher fused to the C-terminal site of HydA.
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We aimed to harness [FeFe] Hydrogenases (HydA)  to accept electrons and therefor enable catalytic production of hydrogen in our project. The optimized coding sequence would increase protein expression in E. coli, theoretically. In the meantime, we conceived to fuse a spycatcher tag to C-terminal of HydA so that the fusion enzyme can be immobilized onto engineered biofilm scaffolds.   
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For more detail information, please visit http://2016.igem.org/Team:ShanghaitechChina/Parts and  http://2016.igem.org/Team:ShanghaitechChina/Hydrogen .
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<partinfo>BBa_K2132004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2132004 SequenceAndFeatures</partinfo>
  
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<div id="Assay" class="content">
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    <div class="col-lg-12">
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        <h2 align="center" style="font-weight:bold">Results of Cloning</h2>
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We fused pACE-Histag-TEV-hydA-Spycatcher with pDK-hydF, pDC-hydE,  pDS-hydG together to give the new plasmid. To test if we successfully fused the four subplasmid, we use single restricted endonuclease digestion of Nde I. The restriction gives three bands on a 1% TAE Gel, in accordance with the band predicted by SnapGene®.
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<center ><img src="https://static.igem.org/mediawiki/2016/7/7a/SHTU_Parts1.png" style="width:70%"></center>
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<p style="text-align:center"><b>Figure 1</b> The test of fusion of the plasmid.</p>
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<p></p>
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<h2 align="center" style="font-weight:bold">Enzyme Activity Assay</h2>
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Unfortunately, compared to the modified HydA protein with SpyTag, the modified HydA protein with SpyCather didn't have the activity of hydrogen production. We will improve or achieve the activity of this modified enzyme in future work.
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<center><img src="https://static.igem.org/mediawiki/2016/d/d8/SHTU_Parts2.png" style="width:70%"></center>
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<p style="text-align:center"><b>Figure 2</b> The Activity of HydA-SpyCatcher results compared with that of HydA-SpyTag.</p>
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</div>
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</div>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 14:13, 21 October 2017


HydA with SpyCatcher, Histag and TEV site

This part encodes the major enzyme of [FeFe] hydrogenase gene clusters (refer to HydA), originally from the bacterium Clostridium acetobutylicum (Original coding sequence: hydA, BBa_K535002, Designed by: iGEM11_UNAM-Genomics_ Mexico.) In particular, this codon-optimized protein was purposely added with Histag and TEV site to the N-terminal site of HydA, and SpyCatcher fused to the C-terminal site of HydA.

We aimed to harness [FeFe] Hydrogenases (HydA) to accept electrons and therefor enable catalytic production of hydrogen in our project. The optimized coding sequence would increase protein expression in E. coli, theoretically. In the meantime, we conceived to fuse a spycatcher tag to C-terminal of HydA so that the fusion enzyme can be immobilized onto engineered biofilm scaffolds.

For more detail information, please visit http://2016.igem.org/Team:ShanghaitechChina/Parts and http://2016.igem.org/Team:ShanghaitechChina/Hydrogen .


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 790
    Illegal AgeI site found at 1300
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2053
    Illegal SapI.rc site found at 663
    Illegal SapI.rc site found at 1005

Results of Cloning

We fused pACE-Histag-TEV-hydA-Spycatcher with pDK-hydF, pDC-hydE, pDS-hydG together to give the new plasmid. To test if we successfully fused the four subplasmid, we use single restricted endonuclease digestion of Nde I. The restriction gives three bands on a 1% TAE Gel, in accordance with the band predicted by SnapGene®.

Figure 1 The test of fusion of the plasmid.

Enzyme Activity Assay

Unfortunately, compared to the modified HydA protein with SpyTag, the modified HydA protein with SpyCather didn't have the activity of hydrogen production. We will improve or achieve the activity of this modified enzyme in future work.

Figure 2 The Activity of HydA-SpyCatcher results compared with that of HydA-SpyTag.