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} | } | ||
+ | @media (min-width: 768px) { | ||
+ | .menu_logo_1 { | ||
+ | top: 18px; | ||
+ | left:10px; | ||
+ | z-index: 1000; | ||
+ | position: fixed; | ||
+ | } | ||
+ | .menu_logo_2 { | ||
+ | top: 18px; | ||
+ | left:100px; | ||
+ | z-index: 1000; | ||
+ | position: fixed; | ||
+ | } | ||
+ | .nav-pills{ | ||
+ | font-size:8px; | ||
+ | font-weight: bold; | ||
+ | } | ||
+ | } | ||
+ | |||
+ | /* 中等屏幕(桌面显示器,大于等于992px) */ | ||
+ | @media (min-width: 992px) { | ||
+ | .menu_logo_1 { | ||
+ | top: 18px; | ||
+ | left:20px; | ||
+ | z-index: 1000; | ||
+ | position: fixed; | ||
+ | } | ||
+ | .menu_logo_2 { | ||
+ | top: 18px; | ||
+ | left:110px; | ||
+ | z-index: 1000; | ||
+ | position: fixed; | ||
+ | } | ||
+ | .nav-pills{ | ||
+ | font-size:10px; | ||
+ | font-weight: bold; | ||
+ | } | ||
+ | } | ||
+ | /* 大屏幕(大桌面显示器,大于等于1200px) */ | ||
+ | @media (min-width: 1200px) { | ||
+ | .menu_logo_1 { | ||
+ | top: 18px; | ||
+ | left:80px; | ||
+ | z-index: 1000; | ||
+ | position: fixed; | ||
+ | } | ||
+ | .menu_logo_2 { | ||
+ | top: 18px; | ||
+ | left:170px; | ||
+ | z-index: 1000; | ||
+ | position: fixed; | ||
+ | } | ||
+ | .nav-pills{ | ||
+ | font-size:12px; | ||
+ | font-weight: bold; | ||
+ | } | ||
+ | } | ||
</style> | </style> | ||
</head> | </head> | ||
− | <body data-spy="scroll" data-target="#myScrollspy" data-offset="20" | + | <body data-spy="scroll" data-target="#myScrollspy" data-offset="20" style="height:auto;"> |
<nav class="navbar navbar-default navbar-fixed-top" style="margin-top:15px"> | <nav class="navbar navbar-default navbar-fixed-top" style="margin-top:15px"> | ||
<div class="container"> | <div class="container"> | ||
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</li> | </li> | ||
<li class="dropdown"> | <li class="dropdown"> | ||
+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown"> | ||
+ | WETLAB <b class="caret"></b> | ||
+ | </a> | ||
+ | <ul class<nav class="navbar navbar-default navbar-fixed-top" style="margin-top:15px"> | ||
+ | <div class="container"> | ||
+ | |||
+ | <div class="navbar-header"> | ||
+ | <a href="https://2017.igem.org/Team:HUST-China" class="navbar-brand logo menu_logo_1"><img src="https://static.igem.org/mediawiki/2017/6/64/Team_HUST_China_2017_logo.png" alt="HUST_iGEM_2017_logo"></a> | ||
+ | <a href="https://2017.igem.org" class="navbar-brand logo menu_logo_2"><img src="https://static.igem.org/mediawiki/2017/f/f6/IGEM_2017_logo.png" alt="HUST_iGEM_2017_logo"></a> | ||
+ | <button type="button" class="navbar-toggle" data-toggle="collapse" data-target="#navbar-collapse" style="background-color: #333; color:#111"> | ||
+ | <span class="icon-bar"></span> | ||
+ | <span class="icon-bar"></span> | ||
+ | <span class="icon-bar"></span> | ||
+ | <span class="icon-bar"></span> | ||
+ | </button> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="collapse navbar-collapse" id="navbar-collapse"> | ||
+ | <ul class="nav navbar-nav navbar-right"> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China">HOME</a></li> | ||
+ | <li class="dropdown"> | ||
+ | <a href="#" class="dropdown-toggle" data-toggle="dropdown"> | ||
+ | PROJECT <b class="caret"></b> | ||
+ | </a> | ||
+ | <ul class="dropdown-menu"> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/project/background">Background</a></li> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/Description">Description</a></li> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/Safety">Safety</a></li> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/Demonstrate">Demonstrate</a></li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="dropdown active"> | ||
<a href="#" class="dropdown-toggle" data-toggle="dropdown"> | <a href="#" class="dropdown-toggle" data-toggle="dropdown"> | ||
WETLAB <b class="caret"></b> | WETLAB <b class="caret"></b> | ||
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</li> | </li> | ||
<li class="dropdown"> | <li class="dropdown"> | ||
− | + | <a href="#" class="dropdown-toggle" data-toggle="dropdown"> | |
− | PARTS | + | PARTS<b class="caret"></b> |
</a> | </a> | ||
+ | <ul class="dropdown-menu"> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/Basic part">Basic part</a></li> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/Composite part">Composite part</a></li> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/Part collection">Part collection</a></li> | ||
+ | </ul> | ||
</li> | </li> | ||
<li class="dropdown"> | <li class="dropdown"> | ||
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<li><a href="https://2017.igem.org/Team:HUST-China/Collaborations"> Collaborations</a></li> | <li><a href="https://2017.igem.org/Team:HUST-China/Collaborations"> Collaborations</a></li> | ||
<li><a href="https://2017.igem.org/Team:HUST-China/HP/Silver">HP/Silver</a></li> | <li><a href="https://2017.igem.org/Team:HUST-China/HP/Silver">HP/Silver</a></li> | ||
+ | <li><a href="https://2017.igem.org/Team:HUST-China/Engagement">Engagement</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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<ul class="nav nav-pills nav-stacked" style="font-size:12px; text-align:center; background:#ccc;"> | <ul class="nav nav-pills nav-stacked" style="font-size:12px; text-align:center; background:#ccc;"> | ||
<li class="active"><a href="#section1">Sensing part</a></li> | <li class="active"><a href="#section1">Sensing part</a></li> | ||
− | <li><a href="#section2"> | + | <li><a href="#section2">Enrichment part<br> and recycling part</a></li> |
<li><a href="#section3">The whole circuit </a></li> | <li><a href="#section3">The whole circuit </a></li> | ||
+ | <li><a href="#section4">Detection of <br>sensing part</a></li> | ||
+ | <li><a href="#section5">Detection of adsorption<br>capacity of LBTs </a></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
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<h4><strong>①Amplify</strong> </h4> | <h4><strong>①Amplify</strong> </h4> | ||
<p> | <p> | ||
− | + | <I><I><I><I><I><I><I>PmrA-PmrB(Fe)</I></I></I></I></I></I></I>、 <I> <I> <I> <I> PmrC-GFP </I></I></I></I> gene through the pcr protocol. | |
</p> | </p> | ||
<h4><strong>②Plasmid Construction</strong> </h4> | <h4><strong>②Plasmid Construction</strong> </h4> | ||
<p> | <p> | ||
− | Coding sequences | + | Coding sequences <I> PmrA-PmrB(Fe)</I> and <I> <I> <I> <I> PmrC-GFP </I></I></I></I>were cloned into the expression vector pBAD30 by restriction enzyme digestion and DNA ligation. |
</p> | </p> | ||
<div class="experiment_circuit col-md-4 col-xs-4 col-md-offset-4 col-xs-offset-4"> | <div class="experiment_circuit col-md-4 col-xs-4 col-md-offset-4 col-xs-offset-4"> | ||
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<h4><strong>③Detection</strong> </h4> | <h4><strong>③Detection</strong> </h4> | ||
<p> | <p> | ||
− | Arabinose was used to induce expression of | + | Arabinose was used to induce expression of PmrA-PmrB-PmrC system. |
</p> | </p> | ||
<p> | <p> | ||
− | We | + | We used the original <I> <I> PmrA-PmrB(Fe) </I> </I>-<I> <I> <I> <I> PmrC-GFP </I></I></I></I>circuit to test whether our |
− | + | PmrA-PmrB-PmrC system works in our plasmid, then the expression and efficacy | |
− | of LBT1-12 were tested . | + | of <I>LBT1-12</I> were tested . |
</p> | </p> | ||
</div> | </div> | ||
− | <div | + | <div style="padding: 10px 100px; text-align: center; font-size:14px;"> |
<table class="table table-hover"> | <table class="table table-hover"> | ||
<thead> | <thead> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(Fe)-PmrC-GFP</I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
− | <td>Test whether our | + | <td>Test whether our PmrABC system works in our plasmid</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT1)-PmrC-GFP</I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
<td> Detecting effectiveness of protein LBT1</td> | <td> Detecting effectiveness of protein LBT1</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT2)-PmrC-GFP</I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td> Detecting effectiveness of protein LBT2</td> | <td> Detecting effectiveness of protein LBT2</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT3)-PmrC-GFP</I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT3</td> | <td>Detecting effectiveness of protein LBT3</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT4)-PmrC-GFP</I></td> |
<td> pBAD30 </td> | <td> pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT4</td> | <td>Detecting effectiveness of protein LBT4</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT5)-PmrC-GFP</I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
<td>Detecting effectiveness of protein LBT5</td> | <td>Detecting effectiveness of protein LBT5</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT6)-PmrC-GFP</I></td> |
<td>pBAD30</td> | <td>pBAD30</td> | ||
<td> Detecting effectiveness of protein LBT6</td> | <td> Detecting effectiveness of protein LBT6</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT7)-PmrC-GFP</I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td> Detecting effectiveness of protein LBT7</td> | <td> Detecting effectiveness of protein LBT7</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT8)-PmrC-GFP</I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT8</td> | <td>Detecting effectiveness of protein LBT8</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT9)-PmrC-GFP</I></td> |
<td> pBAD30 </td> | <td> pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT9</td> | <td>Detecting effectiveness of protein LBT9</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT10)-PmrC-GFP</I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td> Detecting effectiveness of protein LBT10</td> | <td> Detecting effectiveness of protein LBT10</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT11)-PmrC-GFP</I></td> |
<td>pBAD30 </td> | <td>pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT11</td> | <td>Detecting effectiveness of protein LBT11</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td><I>PmrA-PmrB(LBT12)-PmrC-GFP</I></td> |
<td> pBAD30 </td> | <td> pBAD30 </td> | ||
<td>Detecting effectiveness of protein LBT12</td> | <td>Detecting effectiveness of protein LBT12</td> | ||
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<div id="section2" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> | <div id="section2" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> | ||
− | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;"> | + | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Enrichment part and recycling part </h3> |
− | <p> | + | <p><strong>The Capture part is to capture the rare earth ions in the environment and to anchor our cells over the silicon net.</strong></p> |
− | + | <h4><strong>①With appropriate primers, PCR was carried out to amplify target gene <I>oprF-LBT1-12</I> and <I>oprF-Sitag</I>.</strong> </h4> | |
− | + | <h4 class="col-xs-12"><strong>②Then we constructed two vectors to achieve expression of <I>LBT</I> and <I>Sitag</I>.</strong> </h4> | |
− | <h4><strong>①With appropriate primers, PCR was carried out to amplify target gene | + | <img title="demo1" src="https://static.igem.org/mediawiki/2017/7/73/2017_HUST_China_Experiment_image2.png" alt="demo1" class="col-xs-4 col-xs-offset-1" style="padding: 10px 0px;"> |
− | < | + | <img title="demo1" src="https://static.igem.org/mediawiki/2017/7/78/2017_HUST_China_Experiment_image3.png" alt="experiment_circuit" alt="demo1" class="col-xs-4 col-xs-offset-2" style="padding: 10px 0px;"> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<p class="col-xs-12"> | <p class="col-xs-12"> | ||
− | We cloned the sequence of | + | We cloned the sequence of <I>oprF-LBT</I> and <I>Sitag</I> into the vector pET28α in order to make the sequence be tagged N-terminally with a 6×His tag. 6×His tag is an affinity tag which allows the tagged recombinant protein to be purified in the process of affinity purification. |
</p> | </p> | ||
<h4 class="col-xs-12"><strong>③Detection</strong> </h4> | <h4 class="col-xs-12"><strong>③Detection</strong> </h4> | ||
<p class="col-xs-12"> | <p class="col-xs-12"> | ||
− | In the meanwhile,we added the FLAG tag to | + | In the meanwhile, we added the FLAG tag to test whether the <I>LBTs</I> and the <I>Sitag</I> was expressed on the cell membrane. |
</p> | </p> | ||
</div> | </div> | ||
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<h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3> | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3> | ||
<p> | <p> | ||
− | + | <I> PmrA-PmrB(Fe)-PmrC-GFP</I> All of our part should be built in one plasmid. Therefore, we use endonuclease and ligase to replace the <I>GFP</I> by <I>oprF-LBT</I> and <I>oprF-Sitag</I>. | |
</p> | </p> | ||
− | + | <img title="demo1" src="https://static.igem.org/mediawiki/2017/5/58/2017_HUST_China_Experiment_image4.png" alt="experiment_circuit" alt="demo1" class="col-xs-4 col-xs-offset-4" style="padding: 10px 0px;"> | |
− | + | ||
− | + | ||
</div> | </div> | ||
+ | |||
+ | <div id="section4" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> | ||
+ | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Detection of sensing part </h3> | ||
+ | <p>We use our medium containing a small amount of rare earth ions to culture our E. coli. When our engineered E. coli sense the existence of rare earth ions, the PmrC promoter will activate the transcription of the <I>GFP</I> gene. Through the detection of <I>GFP</I> expression, we can know the efficacy of the sensing part. </p> | ||
+ | </div> | ||
+ | |||
+ | <div id="section5" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> | ||
+ | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Detection of adsorption capacity of LBTs </h3> | ||
+ | <p class="col-xs-12">A method of precipitation titration is used to detect the level of rare earth ions adsorption. We add a certain concentration of bacteria to the solution containing different concentrations of rare earth ions. Then centrifuge to remove the bacteria in the solution. | ||
+ | </p> | ||
+ | |||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/1/1a/2017_HUST_China_Experiment_image5.png" alt="experiment_circuit" alt="demo1" class="col-xs-4 col-xs-offset-4" style="padding: 10px 0px;"> | ||
+ | |||
+ | <p class="col-xs-12">In order to make the terbium ions sufficiently precipitated, we add excess base. Rinse the precipitate and dissolve the precipitate with 0.1 M hydrochloric acid titration. In this way, we can know the amount of terbium ions which have already adsorbed on the surface of the bacteria. | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
</div> | </div> | ||
+ | |||
<nav class="col-sm-2" id="myScrollspy2.0"> | <nav class="col-sm-2" id="myScrollspy2.0"> | ||
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</div> | </div> | ||
</footer> | </footer> | ||
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</html> | </html> |
Latest revision as of 09:09, 15 December 2017