Difference between revisions of "Team:ShanghaitechChina/Parts"

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{{ShanghaitechChina}}
 
{{ShanghaitechChina}}
 
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<img class="imgnav" src="https://static.igem.org/mediawiki/2016/3/32/T--ShanghaitechChina--member--bf--Parts.png">
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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
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<a href="#p1">Introduction</a>
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<a href="#p2">Basic Parts</a>
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<a href="#p3">Part Collection</a>
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<a href="#p4">Reference</a>
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          <h1 align="center">Introduction</h1>
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            For our project, we decided to submit seven BioBricks to the iGEM Registry in the pSB1C3 plasmid backbone. Our submitted parts can be divided into two separate collections (Named as Collection One and Collection Two). Collection One is associated with Engineered biofilm subunit CsgA, while Collection Two is relevant to construction of hydrogenase gene clusters.
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          <p></p>
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          All of the constructs are protein-coding parts have been confirmed with gene sequences, demonstrated with expected functionalities, and thus it would be easy to handle for other users. Specifically, in order to make hydrogenase expression more productive, we optimized the gene sequences of 5 hydrogenases parts with the help of OptimumGene™ (GenScript). We changed the codon usage bias in <em>E. coli</em> by upgrading the CAI (Codon Adaptation Index) from a low level (around 0.30) to 0.97.
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            <p></p>
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            Characterizations of the parts are included on the webpage of the parts section, but for more information about the submitted parts in real use, please go to <a href="https://2016.igem.org/Team:ShanghaitechChina/Biofilm">Biofilm</a> or <a href="https://2016.igem.org/Team:ShanghaitechChina/Hydrogen">Hydrogen</a> page.
  
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<h5>Note</h5>
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
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          <h1 align="center">Basic Parts</h1>
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<th><strong>Name</strong></th>
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<th><strong>Type</strong></th>
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<th><strong>Description</strong></th>
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<th><strong>Designer</strong></th>
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<th><strong>Length</strong></th>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132001">BBa_K2132001</a></td>
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<td>Coding</td>
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<td>CsgASpyCatcherHisTag</td>
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<td>Shijie Gu</td>
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<td>867</td>
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</tr>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132003">BBa_K2132003</a></td>
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<td>Coding</td>
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<td>mCherry-SpyTag</td>
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<td>Shijie Gu</td>
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<td>813</td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132004">BBa_K2132004</a></td>
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<td>Coding</td>
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<td>HydA with SpyCatcher, Histag and TEV site</td>
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<td>Yifan Chen</td>
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<td>2166</td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132005">BBa_K2132005</a></td>
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<td>Coding</td>
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<td>HydA with SpyTag, Histag and TEV site</td>
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<td>Yifan Chen</td>
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<td>1857</td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132006">BBa_K2132006</a></td>
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<td>Coding</td>
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<td>HydE with Histag and TEV site</td>
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<td>Yifan Chen</td>
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<td>1098</td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132007">BBa_K2132007</a></td>
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<td>Coding</td>
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<td>HydF with Histag and TEV site</td>
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<td>Yifan Chen</td>
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<td>1281</td>
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<td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132008">BBa_K2132008</a></td>
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<td>Coding</td>
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<td>HydG with Histag and TEV site</td>
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<td>Yifan Chen</td>
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<td>1464</td>
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</table>
  
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<h5>Adding parts to the registry</h5>
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<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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        <h1 align="center">Part Collection</h1>
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        <h4>1.Parts Collection One (Engineered Biofilm Subunit CsgA with SpyCatcher and HisTag)</h4>
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a) In order to realize covalent link of two proteins, we make use of SpyTag and SpyCatcher system (see our Linkage System on Biofilm Page). The SpyTag is fused to HydA of the hydrogenase system, thus a SpyCatcher on CsgA would enable Spy-tagged Hydrogenase to be covalently attached to CsgA In the meantime, to endow biofilm CsgA protein with additional functionalities (such as specific binding to QDs or Nanorods in our case), we append one or two HisTag  at the N- or/and C-terminus of the CsgA-spycatcher protein.  This lead to two new constructs, HisTag-CsgA-SpyCatcher-HisTag (<a href="http://parts.igem.org/Part:BBa_K2132001">BBa_K2132001</a>) and HisTag-CsgA-SpyCatcher (the former being <a href="https://static.igem.org/mediawiki/2016/a/aa/BBa_K2132001.pdf">sequence confirmed</a> and submitted).
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➤ CsgASpyCatcherHisTag (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132001">BBa_K2132001</a>
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➤ mCherry-SpyTag (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132003">BBa_K2132003</a>
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b) To determine if  expression of the two proteins is successful, we constructed mCherry-SpyTag to test the activity of the SpyCatcher on the CsgA. mCherry-SpyTag is thus submitted as <a href="http://parts.igem.org/Part:BBa_K2132003">BBa_K2132003</a>, sequence confirmed.
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      <h4>2.Parts Collection Two (Hydrogenase gene clusters)</h4>
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We optimized [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (optimized coding sequence: hydA, <a href="http://parts.igem.org/Part:BBa_K2132004">BBa_K2132004</a> & <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>) to accept electrons and therefor enable catalytic production of hydrogen in our project. Synthesis of heterologous [FeFe] hydrogenase in <em>E. coli</em> requires co-expression of HydE (optimized coding sequence: hydE, <a href="http://parts.igem.org/Part:BBa_K2132006">BBa_K2132006</a>), HydF (optimized coding sequence: hydF, <a href="http://parts.igem.org/Part:BBa_K2132007">BBa_K2132007</a>), and HydG (optimized coding sequence: hydG, <a href="http://parts.igem.org/Part:BBa_K2132008">BBa_K2132008</a>).
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In this collection, we sub-cloned the coding sequence into the pSB1C3 individually, with two appended tags at the N-terminus (His-tag to faciliate purification and TEV site as cleavable site for Histag cutting off). We have confirmed that the presence of the two tags won’t disrupt expression and normal functionalites of HydA.
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➤ HydA with SpyCatcher, Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132004">BBa_K2132004</a>
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➤ HydA with SpyTag, Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>
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<p></p>
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➤ HydE with Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132006">BBa_K2132006</a>
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<p></p>
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➤ HydF with Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132007">BBa_K2132007</a>
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<p></p>
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➤ HydG with Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132008">BBa_K2132008</a>
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<h6>★Optimization of this collection</h6>
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<h6>The original sequences of hydrogenase were found in <a href="www.genome.jp">www.genome.jp</a>. With the help of OptimumGene™, We used the following parameters to optimize our gene sequences without changing their amino acids sequence: Codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, Cryptic splicing sites, Premature PolyA sites, Internal chi sites and ribosomal binding sites, Negative CpG islands, RNA instability motif (ARE), Repeat sequences (direct repeat, reverse repeat, and Dyad repeat).</h6>
  
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<h5>What information do I need to start putting my parts on the Registry?</h5>
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<p>The information needed to initially create a part on the Registry is:</p>
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
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<h5>Inspiration</h5>
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<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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            King P W, Posewitz M C, Ghirardi M L, et al. Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System[J]. Journal of Bacteriology, 2006, 188(6):2163-72.
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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<groupparts>iGEM2016 Example</groupparts>
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Latest revision as of 15:14, 19 October 2016

igem2016:ShanghaiTech