Difference between revisions of "Team:Pasteur Paris/Description"

 
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<h1><B>Parts description</B></h1>
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<h3>BBa_K2053000  (for Bronze medal): <i>Thalassiosira pseudonana</i> silaffin kinase (tpSTK1)</h3>
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<i>T. pseudonana</i> silaffin kinase (tpSTK1) is a central BioBrick part. It was described as an efficient diatom silaffin-phosphorylating enzyme [1]. Since it was shown that phosphorylation of silaffins increased their biosilification activity [2], we hypothesized that phosphorylation of silica-binding peptide from our fusion protein would increase its biosilification properties.</br></br>
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To facilitate protein purification, we added a HisTag. To remove the HisTag, we separated the HisTag from the sequence by the TEV cleavage site sequence. </br>
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<h4>BBa_K2053002 (for Silver medal) : Fusion Si4-cellulose-binding domain-B of domain protein A</h4>
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In order to functionalize a cellulose-based patch into a biodetection device we designed and created a fusion protein by assembling three parts: the phage displayed silica-binding peptide (Si4) [3],  the <i>Clostridium cellulovorans</i> cellulose-binding domain of cellulose-binding protein A (CBPa) [4], and the B domain of <i>Staphylococcus aureus</i> protein A (BpA) [5]. This fusion protein is used to bind to cellulose-based patch, increase its rigidity by biosilification, and make it a detection device by fixing the Fc fragment of specific antibodies. We used the preexisting iGEM BioBrick parts that we assembled thanks to commonly used flexible linkers [6]. </br></br>
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To facilitate protein purification, we added a His-Tag composed  of 6 histidines at the N-terminus, or C-terminus. To remove the His-Tag, we inserted a TEV (Tobacco Etch virus) cleavage site downstream from the His-Tag. </br></br>
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<h5> Fusion cellulose-binding domain of CBP-B domain of protein A (for Gold medal)</h5>
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We improved previously existing BBa_K863110 and BBa_K103003 BioBricks parts by combining them, creating one part. It is a fusion protein between the <i>Clostridium cellulovorans</i> cellulose-binding domain of cellulose-binding protein A (CBPa) with the B domain of <i>Staphylococcus aureus</i> protein A (BpA). This fusion protein can be used to bind antibodies to cellulose.</br></br>
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To facilitate protein purification, we added a His-Tag. To remove the His-Tag, we separated the HisTag from the sequence by the TEV cleavage site sequence.</br>
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<h2>References: </h2>
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[1] Characterization of an Endoplasmic Reticulum-associated Silaffin Kinase from the diatom <i>Thalassiosira pseudonana</i>, Sheppard V et al, 2010, The Journal of Biological Chemistry</br>
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[2] The Role of Proteins in Biosilification, Otzen D, 2012, Scientifica</br>
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[3] Silica-Precipitating Peptides Isolated from a Combinatorial Phage Display Peptide Library Naik et al, 2002, Journal of Nanoscience and Nanotechnology</br>
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[4] Characterization of the cellulose-binding domain of the <i>Clostridium cellulovorans</i> cellulose-binding protein A, Goldstein MA, 1993, J Bacteriol</br>
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[5] Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from <i>Staphylococcus aureus</i> at 2.9- and 2.8-A resolution, Deisenhofer J, 1981, Biochemistry</br>
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[6] Fusion Protein Linkers: Property, Design and Functionality, Xiaoying C, 2013, Advanced Drug Delivery Reviews</br>
  
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Latest revision as of 09:34, 14 October 2016