|
|
| (19 intermediate revisions by the same user not shown) |
| Line 1: |
Line 1: |
| − | {{WHU-China}}
| |
| − | <html>
| |
| | | | |
| − | <head></head>
| |
| − | <body>
| |
| − |
| |
| − |
| |
| − | <div id="background">
| |
| − | <img id="background-image" src="https://static.igem.org/mediawiki/2017/6/69/WHU-China-child-bg.jpeg" alt=""/>
| |
| − | </div>
| |
| − | <div id="header">
| |
| − | <div id="nav-wrapper">
| |
| − | <div class="nav">
| |
| − | <ul class="nv">
| |
| − | <li id="nv0" style="width:56px"><a href="#">logo</a></li>
| |
| − | <li id="nv1">
| |
| − | <a>Project</a>
| |
| − | <ul>
| |
| − | <li id="nv11"><a href="#">Description</a></li>
| |
| − | <li id="nv12"><a href="#">Design</a></li>
| |
| − | <li id="nv13"><a href="#">Experiments</a></li>
| |
| − | <li id="nv14"><a href="#">Results</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li id="nv2">
| |
| − | <a>Parts</a>
| |
| − | <ul>
| |
| − | <li id="nv21"><a herf="#">Basic Part</a></li>
| |
| − | <li id="nv22"><a href="#">Composite Part</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li id="nv3"><a href="#">Interlab</a></li>
| |
| − | <li id="nv4"><a href="#">Models</a></li>
| |
| − | <li id="nv5">
| |
| − | <a>Human Practices</a>
| |
| − | <ul>
| |
| − | <li id="nv51"><a href="#">Activities</a></li>
| |
| − | <li id="nv52"><a href="#">Silver</a></li>
| |
| − | <li id="nv53"><a href="#">Gold</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li id="nv6"><a href="#">Safety</a></li>
| |
| − | <li id="nv7">
| |
| − | <a>Team</a>
| |
| − | <ul>
| |
| − | <li id="nv71"><a href="#">Team Members</a></li>
| |
| − | <li id="nv72"><a href="#">Collaborations</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − |
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div id="topbanner-wrapper" style="height: 299.462px;">
| |
| − | <div id="topbanner">
| |
| − | </div>
| |
| − | </div>
| |
| − | <div id="contents-wrapper">
| |
| − | <div id="contents">
| |
| − | <div>
| |
| − | <h6>Description</h6>
| |
| − | <ul id="cont">
| |
| − | <li><a href="#Brief">1 Brief introduction in RdhANP</a></li>
| |
| − | <li><a href="#Description">2 Description of our operating bacteria</a></li>
| |
| − | <li><a href="#Modularization">3 Modularization: Overview</a></li>
| |
| − | <li><a href="#protocol">4 Our protocol</a></li>
| |
| − | <li><a href="#Appendix">5 Appendix</a></li>
| |
| − |
| |
| − | </ul>
| |
| − |
| |
| − |
| |
| − |
| |
| − | <h1 id="Brief">Brief introduction in RdhANP</h1>
| |
| − |
| |
| − | <p>RdhANP is a native protein of Nitratireducror pacificus pht-3B. It is codon-optimized for operated and expression in Bacillus magaterium. RdhANP can catalyze the reduction of ortho-halogenated phenolic compound, which endows it with biological dehalogenation function. Different from other reductive proteins that are membrane-associated and oxygen-sensitive in their family, RdhANP is a soluble cytoplasmic one and oxygen-tolerant, which is fairly good for heterologous expression in other chassis. However, this enzyme displays a strict requirement for o-halogenated phenolic substrates and B12 cofactor; it also should under reductive condition using either reduced methyl viologen or NADPH. </p>
| |
| − |
| |
| − | </div>
| |
| − | <div>
| |
| − | <h1 id="Description">Description of our operating bacteria</h1>
| |
| − |
| |
| − | <p>Nitratireductor pacificus strain pht-3BT (MCCC 1A01024T, CCTCC AB 209302T, LMG 25541T) was isolated from deep sea sediments of the western Pacific Ocean enriched with pyrene as the sole carbon and energy source. Cells were Gram-negative, short rods that were motile by means of flagella. They were first grown on marine agar 2216 (BD) medium, which produces smooth grey non-pigmented colonies with regular edges and slightly raised in the center, 2–3 mm in diameter after 3 days at 28 ℃ .We use MB medium as alternative. The most important feature of the cell is its production of RdhANP. </p>
| |
| − |
| |
| − | </div>
| |
| − | <div>
| |
| − | <h1 id="Modularization">Modularization: Overview</h1>
| |
| − |
| |
| − | <img src="https://static.igem.org/mediawiki/2017/8/88/WHU-China-Modularization-Overview.png" width="850" height="893" alt=""/> </div>
| |
| − |
| |
| − | <h1 id="protocol">Our protocol</h1>
| |
| − | <table>
| |
| − | <tbody>
| |
| − |
| |
| − | <p>First, digest the vector pUC57 and the target fragment psb1C3 with two kinds of endonucleases, e.g. PstⅠand EcoRⅠ. Second, using agarose gel electrophoresis (AGE) to analyze the result of restriction endonuclease digestion. Third, recycle the agarose gel containing target bands to enrich the products. Forth, link the vector and target gene by T4 ligase. Fifth, using AGE again to evaluate the result. Finally, transform the recombination plasmids to the bacillus megatherium.</p>
| |
| − |
| |
| − | <tr>
| |
| − | <td><p>During the process, we encounter some problems. A typical one is that we failed in the transformation step several times. After debugging the experiment conditions in various aspects, we finally found the most appropriate condition. Another reason responsible for it is the threshold –below concentration of target segments contained in the recycled gel.</p></td>
| |
| − | <td><img src="https://static.igem.org/mediawiki/2017/c/c2/WHU-China-Our-protocol.jpeg" width="300" height="150" alt=""/></td>
| |
| − | </tr>
| |
| − |
| |
| − | </tbody></table>
| |
| − | <br><br><br>
| |
| − | <div>
| |
| − | <h1 id="Appendix">Appendix</h1>
| |
| − | <strong>#Gene sequence (Without prefixes and suffixes)</strong>
| |
| − | <p>ATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGA </p>
| |
| − |
| |
| − | <strong>#pSB1C3</strong>
| |
| − | <p>TACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAG </p>
| |
| − |
| |
| − | <strong>#sequencing primer</strong>
| |
| − | <p>senCe
| |
| − | 5' CAGATAAAAAAAATCCTTAG 3'
| |
| − | AnTisenCe
| |
| − | 5' TTCGGTTTTAAAGAAAAAGG 3'
| |
| − | </p>
| |
| − | <strong>#recombinant plasmid</strong>
| |
| − | <p>ATTCGCGGCCGCTTCTAGATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGATAATAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGG
| |
| − | </p>
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − | <!--<h1 id="Brief">Brief introduction in RdhANP</h1>
| |
| − | <table>
| |
| − | <tbody><tr>
| |
| − | <td><strong>#1 Integrated Human Practices</strong><br>Expand on your silver medal activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project.</td>
| |
| − | <td>Faced with the people's overwhelming concerns regarding the application of genetic engineering, we strived to improve upon our project design of Noro-catcher and introduced a new module to increase its safety. We also have conducted a lecture/workshop to students of age 13-18, and observed that their overall acceptance of genetic engineering increased by introducing them to basic knowledge on the matter. Visit <a href="https://2016.igem.org/Team:Kyoto/Integrated_Practices" target="_blank">Integrated Practices</a> and <a href="https://2016.igem.org/Team:Kyoto/HP/Gold" target="_blank">HP/Gold</a> for details. </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong>#2 Improve a previous part or project</strong><br>Improve the function OR characterization of an existing BioBrick Part or Device and enter this information in the Registry. Please see the Registry help page on how to document a contribution to an existing part. This part must NOT be from your 2016 part number range.</td>
| |
| − | <td>We enhanced iGEM12_Penn's INPNC-MCS to INPNC-6xHistag and improved its function. Visit <a href="http://parts.igem.org/Part:BBa_K811005" target="_blank">the original parts page</a> and <a href="http://parts.igem.org/Part:BBa_K1933001" target="_blank">our enhanced parts page</a> for details</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td><strong>#3 Proof of concept</strong><br>Demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept. (biological materials may not be taken outside the lab).</td>
| |
| − | <td>Our ultimate goal was to create <i>E. coli</i> which can bind to NoV, and improved upon surface expression systems INPNC and BclA to achieve this. In detail, we enabled the purification and detection of the fusion protein by the addition of His tag, regardless of the passenger proteins placed downstream. Furthermore, we observed the binding of scFv surface expressing <i>E. coli</i> to NoVLP using SEM. Visit our <a href="https://2016.igem.org/Team:Kyoto/Proof" target="_blank">Proof of concept </a>and <a href="http://parts.igem.org/Part:BBa_K1933200" target="_blank">Parts</a> for details. </td>
| |
| − | </tr>
| |
| − | </tbody></table>
| |
| − | <br><br><br>
| |
| − | -->
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − | <div id="footer">
| |
| − | <!--<div id="footer_logos">
| |
| − | <img src="./Team_Kyoto_Achievements - 2016.igem.org_files/T--Kyoto--Footer-24.png" width="874" height="95">
| |
| − | </div>
| |
| − | <div id="footer_contact">
| |
| − | <img src="./Team_Kyoto_Achievements - 2016.igem.org_files/T--Kyoto--Footer-25.png">
| |
| − | </div>
| |
| − | <div id="footer_social">
| |
| − | <a href="mailto:igemkyoto@gmail.com"><img src="./Team_Kyoto_Achievements - 2016.igem.org_files/T--Kyoto--Footer-28.png"></a>
| |
| − | <a href="https://twitter.com/igemkyoto"><img src="./Team_Kyoto_Achievements - 2016.igem.org_files/T--Kyoto--Footer-26.png"></a>
| |
| − | <a href="https://www.facebook.com/IgemKyoto/"><img src="./Team_Kyoto_Achievements - 2016.igem.org_files/T--Kyoto--Footer-27.png"></a>
| |
| − | </div>-->
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <!--<div id="background">
| |
| − | <img id="background-image" src="child-bg.jpg" alt=""/>
| |
| − | </div>
| |
| − | <div id="header">
| |
| − | <div id="nav-wrapper">
| |
| − | <div class="nav">
| |
| − | <ul class="nv">
| |
| − | <li id="nv0" style="width:56px"><a href="#">logo</a></li>
| |
| − | <li id="nv1">
| |
| − | <a>Project</a>
| |
| − | <ul>
| |
| − | <li id="nv11"><a href="#">Description</a></li>
| |
| − | <li id="nv12"><a href="#">Design</a></li>
| |
| − | <li id="nv13"><a href="#">Experiments</a></li>
| |
| − | <li id="nv14"><a href="#">Results</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li id="nv2">
| |
| − | <a>Parts</a>
| |
| − | <ul>
| |
| − | <li id="nv21"><a herf="#">Basic Part</a></li>
| |
| − | <li id="nv22"><a href="#">Composite Part</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li id="nv3"><a href="#">Interlab</a></li>
| |
| − | <li id="nv4"><a href="#">Models</a></li>
| |
| − | <li id="nv5">
| |
| − | <a>Human Practices</a>
| |
| − | <ul>
| |
| − | <li id="nv51"><a href="#">Activities</a></li>
| |
| − | <li id="nv52"><a href="#">Silver</a></li>
| |
| − | <li id="nv53"><a href="#">Gold</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li id="nv6"><a href="#">Safety</a></li>
| |
| − | <li id="nv7">
| |
| − | <a>Team</a>
| |
| − | <ul>
| |
| − | <li id="nv71"><a href="#">Team Members</a></li>
| |
| − | <li id="nv72"><a href="#">Collaborations</a></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − |
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div id="topbanner-wrapper" style="height: 299.462px;">
| |
| − | <div id="topbanner">
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="column full_size">
| |
| − | <h1>Description</h1>
| |
| − | <h3>Part: BBa_</h3>
| |
| − | <h5>Brief introduction in RdhANP</h5>
| |
| − | <ul>
| |
| − | <li>RdhANP is a native protein of Nitratireducror pacificus pht-3B. It is codon-optimized for operated and expression in Bacillus magaterium. RdhANP can catalyze the reduction of ortho-halogenated phenolic compound, which endows it with biological dehalogenation function. Different from other reductive proteins that are membrane-associated and oxygen-sensitive in their family, RdhANP is a soluble cytoplasmic one and oxygen-tolerant, which is fairly good for heterologous expression in other chassis. However, this enzyme displays a strict requirement for o-halogenated phenolic substrates and B12 cofactor; it also should under reductive condition using either reduced methyl viologen or NADPH.</li>
| |
| − |
| |
| − | </ul>
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − | <div class="column full_size" >
| |
| − |
| |
| − | <h5>Description of our operating bacteria</h5>
| |
| − |
| |
| − | <ul>
| |
| − | <li>Nitratireductor pacificus strain pht-3BT (MCCC 1A01024T, CCTCC AB 209302T, LMG 25541T) was isolated from deep sea sediments of the western Pacific Ocean enriched with pyrene as the sole carbon and energy source. Cells were Gram-negative, short rods that were motile by means of flagella. They were first grown on marine agar 2216 (BD) medium, which produces smooth grey non-pigmented colonies with regular edges and slightly raised in the center, 2–3 mm in diameter after 3 days at 28 ℃ .We use MB medium as alternative. The most important feature of the cell is its production of RdhANP. </li>
| |
| − |
| |
| − | </ul>
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="column half_size" >
| |
| − |
| |
| − | <h5>Modularization: Overview</h5>
| |
| − | <ul>
| |
| − | <li> <a><img src="https://static.igem.org/mediawiki/2017/8/88/WHU-China-Modularization-Overview.png" /></a> </li>
| |
| − |
| |
| − | </ul>
| |
| − |
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <div class="column half_size" >
| |
| − | <h5>Our protocol</h5>
| |
| − |
| |
| − | <ul>
| |
| − | <li>First, digest the vector pUC57 and the target fragment psb1C3 with two kinds of endonucleases, e.g. PstⅠand EcoRⅠ. Second, using agarose gel electrophoresis (AGE) to analyze the result of restriction endonuclease digestion. Third, recycle the agarose gel containing target bands to enrich the products. Forth, link the vector and target gene by T4 ligase. Fifth, using AGE again to evaluate the result. Finally, transform the recombination plasmids to the bacillus megatherium.
| |
| − |
| |
| − | </li>
| |
| − | <li> During the process, we encounter some problems. A typical one is that we failed in the transformation step several times. After debugging the experiment conditions in various aspects, we finally found the most appropriate condition. Another reason responsible for it is the threshold –below concentration of target segments contained in the recycled gel.
| |
| − | <a><img src="https://static.igem.org/mediawiki/2017/c/c2/WHU-China-Our-protocol.jpeg" /></a>
| |
| − | </li>
| |
| − |
| |
| − | </ul>
| |
| − | </div>
| |
| − |
| |
| − | <div class="column half_size" >
| |
| − | <h5>Appendix</h5>
| |
| − |
| |
| − |
| |
| − | <p>Gene sequence (Without prefixes and suffixes)</p>
| |
| − |
| |
| − | <ul>
| |
| − | <li>ATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGA</li>
| |
| − |
| |
| − | </ul>
| |
| − |
| |
| − |
| |
| − |
| |
| − | <p>pSB1C3</p>
| |
| − |
| |
| − | <ul>
| |
| − | <li>TACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAG</li>
| |
| − |
| |
| − | </ul>
| |
| − |
| |
| − |
| |
| − | <p>sequencing primer</p>
| |
| − |
| |
| − | <ul>
| |
| − | <li>senCe</li>
| |
| − | <li>5' CAGATAAAAAAAATCCTTAG 3'</li>
| |
| − | <li>AnTisenCe</li>
| |
| − | <li>5' TTCGGTTTTAAAGAAAAAGG 3'</li>
| |
| − |
| |
| − | </ul>
| |
| − |
| |
| − | <p>recombinant plasmid</p>
| |
| − |
| |
| − | <ul>
| |
| − | <li>ATTCGCGGCCGCTTCTAGATGAGACTTTACAGCAATAGAGATAGACCGAATCATCTTGGCCCGCTGGCGCTGGAAAGACTTGCAAGAGTTGATGATGTGGTGGCACAGCCGGCGAGACAGCCGGAAGATGGCTTTGCAGCGTCAGAAGATTCACTGCTGGGCGATGTTGAAGAATATGCGAGACTTTTTACAAGATTTCTTGATGGACCTGTTGCGCCGCTTGGAGATGCAATTCCGGATGATCCGGCAAGAAGAGCGGAAAATCTGAAAGCATCAGCGTATTTTCTGGATGCGTCAATGGTGGGAATTTGTAGACTTGATCCGGATGATAGAGCGGGAGATTGCGATCCGAGCCATACACATGCACTTGTTTTTGCGGTGCAGTTTGGCAGAGAACCGGAAGCAGGCGAAGCGGGAGCGGAATGGATTAGAGGAACAAATGCGGCAAGAACAGATATGAGATGTGCGGAAATTGCAGCAATTCTTAGCGGATATGTTAGATGGATGGGATTTCCGGCGAGAGGACATTTTAGCGGCGATGCGCAAGTTGATCTGGCAAGACTGGCAGTTAGAGCAGGCCTGGCGAGAGTTGTGGATGGCGTGCTGGTTGCACCGTTTCTGAGAAGAGGCTTTAGACTGGGAGTGGTTACAACAGGATATGCGCTTGCAGCAGATAGACCGCTGGCGCCGGAAGGAGATCTGGGCGAAACAGCGCCGGAAGTTATGCTGGGAATTGATGGCACAAGACCGGGCTGGGAAGATGCGGAAGAAGAAAAAAGACCGCTGCATATGGGCAGATATCCGATGGAAACAATTAGAAGAGTTGATGAACCGACAACACTTGTGGTGAGACAGGAAATTCAGAGAGTTGCAAAAAGAGGCGATTTCTTTAAAAGAGCAGAAGCAGGCGATCTGGGCGAGAAAGCAAAACAGGAAAAGAAAAGATTTCCGATGAAACATCCGCTGGCACTGGGCATGCAACCGCTGATTCAGAATATGGTGCCGCTTCAAGGAACAAGAGAAAAACTGGCGCCGACAGGAAAAGGAGGCGATCTTAGCGATCCGGGCAGAAATGCGGAAGCAATTAAGGCACTGGGCTATTATCTGGGCGCAGATTTTGTGGGAATTTGCAGAGCAGAACCGTGGATGTATTATGCGAGCGATGAAGTGGAAGGCAAACCGATTGAAGCATATCATGATTATGCGGTTGTGATGCTGATTGATCAGGGATATGAAACAATGGAAGGCGCGAGCGGCGATGATTGGATTTCAGCAAGCCAATCAATGAGAGCGTATATGAGAGGAGCAGAAATTGCGGGAGTTATGGCAGCACATTGTAGAAGAATGGGATATTCAGCGAGAAGCCATAGCAATGCACATTCAGAAGTTATTCATAATCCGGCGATTCTTATGGCAGGACTGGGAGAAGTGAGCAGAATTGGAGATACACTTCTTAATCCGTTTATTGGCCCGAGATCAAAAAGCATTGTTTTTACAACAGACCTTCCGATGAGCGTGGATAGACCGATTGATTTTGGCCTGCAAGATTTTTGTAATCAATGCAGAAAATGCGCAAGAGAATGTCCGTGTAATGCAATTAGCTTTGGCGATAAAGTTATGTTTAACGGATATGAAATCTGGAAAGCAGATGTGGAAAAATGCACAAAATATAGAGTTACACAGATGAAAGGCTCAGCGTGTGGCAGATGTATGAAAATGTGCCCGTGGAATAGAGAAGATACAGTGGAAGGCAGAAGACTGGCAGAACTTTCAATTAAGGTGCCGGAAGCAAGAGCAGCAATTATTGCAATGGATGATGCACTGCAAAATGGCAAAAGAAATCTGATTAAGAGATGGTGGTTTGATCTTGAAGTGATTGATGGAGTTGCAGGCGCGCCGAGAATGGGAACAAATGAAAGAGATCTTTCACCGGATAGAGGAGATAAAATTGGAGCAAATCAAAAACTTGCAATGTATCCGCCGAGACTTCAACCGCCGCCGGGAACAACACTTGATGCGGTTCTTCCGGTTGATAGAAGCGGAGGCCTTGCAGAATATGCGGCGGCAGAAACACCGGCGGCAGCAAGAGCAAGACTTAAATCATCAGCGGGATAATAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGG</li>
| |
| − |
| |
| − | </ul>
| |
| − | </div>-->
| |
| − |
| |
| − | </body>
| |
| − |
| |
| − | </html>
| |
