|
|
| Line 85: |
Line 85: |
| | <li> Group: ShanghaitechChina</li> | | <li> Group: ShanghaitechChina</li> |
| | <li> Author: Yifan Chen</li> | | <li> Author: Yifan Chen</li> |
| − | <li> Summary: We optimized [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (Original coding sequence: hydA, <a href="http://parts.igem.org/Part:BBa_K535002">BBa_K535002</a>. Optimized coding sequence: hydA with SpyTag and Histag <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. The optimized coding sequence would produce more protein </li> | + | <li> Summary: We optimized [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (Original coding sequence: hydA, <a href="http://parts.igem.org/Part:BBa_K535002">BBa_K535002</a>. Optimized coding sequence: hydA with SpyTag and Histag <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. The optimized coding sequence would produce more protein, theoretically. And optimization also improved the activity of [FeFe] Hydrogenases according to the experiment that we did.</li> |
| | </ul></h4> | | </ul></h4> |
| | <h3>>Improvement</h3> | | <h3>>Improvement</h3> |
| − | <h4>Quantum dots binding test</h4> | + | <h4>Hydrogenases expression and enzyme activity assay</h4> |
| | | | |
| | | | |
| Line 100: |
Line 100: |
| | | | |
| | | | |
| − | <div id="p2" class="content">
| |
| − | <div class="row">
| |
| − | <div class="col-lg-12">
| |
| − | <h1 align="center">Optimize the codon</h1>
| |
| − | </div>
| |
| − |
| |
| − | <div class="col-lg-12">
| |
| − | <h4>2.Parts Collection Two (Hydrogenase gene clusters)</h4>
| |
| − | We utilize [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (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 (coding sequence: hydE, <a href="http://parts.igem.org/Part:BBa_K2132006">BBa_K2132006</a>), HydF (coding sequence: hydF, <a href="http://parts.igem.org/Part:BBa_K2132007">BBa_K2132007</a>), and HydG (coding sequence: hydG, <a href="http://parts.igem.org/Part:BBa_K2132008">BBa_K2132008</a>).
| |
| − | <p></p>
| |
| − | 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.
| |
| − | <p></p>
| |
| − | ➤ HydA with SpyCatcher, Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132004">BBa_K2132004</a>
| |
| − | <p></p>
| |
| − | ➤ HydA with SpyTag, Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>
| |
| − | <p></p>
| |
| − | ➤ HydE with Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132006">BBa_K2132006</a>
| |
| − | <p></p>
| |
| − | ➤ HydF with Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132007">BBa_K2132007</a>
| |
| − | <p></p>
| |
| − | ➤ HydG with Histag and TEV site (<em>E. coli</em>) - <a href="http://parts.igem.org/Part:BBa_K2132008">BBa_K2132008</a>
| |
| − | <h6>★Optimization of this collection</h6>
| |
| − | <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>
| |
| | | | |
| | </div> | | </div> |
