|
|
| Line 1: |
Line 1: |
| - | <html>
| |
| - | </div>
| |
| - | </div>
| |
| - | </div>
| |
| | | | |
| - |
| |
| - | <style type="text/css">
| |
| - | *{margin:0;padding:0;font-family:"微软雅黑","Arial";}
| |
| - |
| |
| - | body{
| |
| - | width: 960px;
| |
| - | height: auto;
| |
| - | margin: 0 auto;
| |
| - | font-size:12px;
| |
| - | font-family:Arial, Helvetica, sans-serif;
| |
| - | background-color:#fff;
| |
| - | }
| |
| - |
| |
| - | h1,h2,h3,h4,h5,h6{
| |
| - | border:none;}
| |
| - |
| |
| - | p {margin:0.5em 0 !important;
| |
| - | text-decoration:none;
| |
| - | font-family:Arial, Helvetica, sans-serif;
| |
| - | font-size:12px;
| |
| - | }
| |
| - | .content-section{
| |
| - | width:960px;
| |
| - | height:10px;
| |
| - | margin-top:0px;
| |
| - | }
| |
| - | .logo-section{
| |
| - | height:220px;
| |
| - | width:960px;
| |
| - | position:relative;
| |
| - | float:left;
| |
| - | margin-top:5px;
| |
| - | background:url(https://static.igem.org/mediawiki/igem.org/1/16/Tju-Project-banner.jpg) no-repeat;
| |
| - | }
| |
| - | .note{
| |
| - | width:960px;
| |
| - | height:200px;
| |
| - | float:left;
| |
| - | margin-top:10px;
| |
| - | position:relative;
| |
| - | background-color:#fff;
| |
| - | padding:0;
| |
| - | }
| |
| - | .note1{
| |
| - | width:960px;
| |
| - | min-height:200px;
| |
| - | float:left;
| |
| - | background-color:#fff;
| |
| - | margin-top:0px;
| |
| - | }
| |
| - | .note2{
| |
| - | width:880px;
| |
| - | height:auto;
| |
| - | float:left;
| |
| - | position:relative;
| |
| - | margin-left:20px;
| |
| - | padding:30px 10px 10px 30px;
| |
| - | }
| |
| - | #footer{
| |
| - | margin:0 auto;
| |
| - | width:960px;
| |
| - | min-height:73px;
| |
| - | text-align:center;
| |
| - | color:#666;
| |
| - | float:left;
| |
| - | }
| |
| - | #goTopBtn {POSITION: fixed;TEXT-ALIGN: center;LINE-HEIGHT: 30px;WIDTH: 100px;BOTTOM: 35px;HEIGHT: 100px;FONT-SIZE: 12px;RIGHT: 30px;}
| |
| - |
| |
| - |
| |
| - | <!--hidden section-->
| |
| - | .hidden {display:none;}
| |
| - | #p-logo,#footer-box {display:none;}
| |
| - | #top-section {border:none;height:15px;}
| |
| - | .firstHeading {display:none;}
| |
| - | #bodyContent{display:none;}
| |
| - | #menubar a{text-decoration:none;color:#CCC;list-style: none;}
| |
| - | #menubar a:hover{text-decoration:none;color:#FFF;list-style: none;}
| |
| - | #search-controls{display:none;}
| |
| - | .printfooter{display:none;}
| |
| - | #catlinks{display:none;}
| |
| - | #content{background:none;padding:0px;margin-top:0px;border:none;}
| |
| - | #toc{display:none;}
| |
| - | </style>
| |
| - |
| |
| - | <!--content section-->
| |
| - |
| |
| - | <style type="text/css">
| |
| - | .sddm{z-index: 30;width: 960px;height:65px;position:relative;float:left;background-color:#FFF;position:raletive;margin-left:0px;}
| |
| - | .sddm ul{margin-left:0px;}
| |
| - | .sddm li{margin: 0;list-style: none;float: left;font: bold 14px arial;height:25px;background-color:#FFF;}
| |
| - | .sddm li a{display: block;margin:0;padding: 15px 0px 15px 0px;width: 96px;background: #FFF;color: #000;text-align: center;text-decoration: none;border-top:#ececec 5px solid;}
| |
| - | .sddm li a:hover{background:#FCFCFC;color:#0babe7;border-top:#0babe7 5px solid;}
| |
| - | .sddm div{position: absolute;width:96px;z-index:999;visibility: hidden;margin: 0;padding: 0;}
| |
| - | .sddm div a{position: relative;display: block;margin: 0;padding: 5px 5px;width: auto;white-space: nowrap;text-align:center;text-decoration: none;color: #000;font: 12px arial}
| |
| - | .sddm div a:hover{background:#FAFAFA;color: #0babe7;}
| |
| - | </style>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="cont1">
| |
| - | <ul class="sddm">
| |
| - |
| |
| - | <style type="text/css">
| |
| - | .A-C{font-size:12px !important;padding-top:0 !important;text-align:center;line-height:100%}
| |
| - | .HP{font-size:14px !important;padding-top:0 !important;text-align:center;}
| |
| - | </style>
| |
| - |
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin" style="padding:20px 0px 15px 0px;height:30px;">Home</a>
| |
| - | </li>
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin/Project" style="padding:20px 0px 15px 0px;height:30px;">Project</a>
| |
| - | </li>
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin/Safety " style="padding:20px 0px 15px 0px;height:30px;">Safety</a>
| |
| - | </li>
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin/Modeling" style="padding:20px 0px 15px 0px;height:30px;">Modeling</a>
| |
| - | </li>
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin/Protocol" style="padding:20px 0px 15px 0px;height:30px;">Protocol</a>
| |
| - | </li>
| |
| - | <li class="HP"><a href="https://2013.igem.org/Team:Tianjin/Human Practice" style="padding:14px 0px 13px 0px;">Human Practice</a>
| |
| - | </li>
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin/Data" style="padding:20px 0px 15px 0px;height:30px;">Data</a>
| |
| - | </li>
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin/Notebook" style="padding:20px 0px 15px 0px;height:30px;">Notebook</a>
| |
| - | </li>
| |
| - | <li><a href=" https://2013.igem.org/Team:Tianjin/Team" style="padding:20px 0px 15px 0px;height:30px;">Team</a>
| |
| - | </li>
| |
| - | <li class="A-C"><a href="https://2013.igem.org/Team:Tianjin/Attributions" style="padding:7px 0px 7px 0px;">Attributions<br/>&<br/>Contributions</a>
| |
| - | </li>
| |
| - | </ul>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="content-section">
| |
| - | </div>
| |
| - |
| |
| - | <div class="logo-section">
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="note1">
| |
| - | <style type="text/css">
| |
| - | .box{ width:960px; margin:0 auto; overflow:hidden;}
| |
| - | .main{ width:720px; height:auto; float:right;position:relative;padding:10px 10px 10px 10px;}
| |
| - | .fixed{ width:220px; height:400px;text-align:center;float:left;word-spacing:0.1em;top:10px;margin-top:10px;}
| |
| - | .main img{border:hidden;margin-bottom:5px;}
| |
| - | .main div,li,p{font-family:Arial;line-height:150%;word-spacing:0.1em;}
| |
| - |
| |
| - |
| |
| - | .box p{color:#000;font-family:Arial, Helvetica, sans-serif;font-size:14px;line-height:150%;text-align:left; clear:both;}
| |
| - | .box h1{text-decoration:none;font-weight:normal;color:#0babe7;font-family:Arial;}
| |
| - | .img1{margin:0 150px 15px 150px;padding:5px 5px 5px 5px;background-color:#fafafa;border:thin solid #999; vertical-align:middle;width:400px;}
| |
| - | .img1 a{target="_blank";}
| |
| - |
| |
| - |
| |
| - |
| |
| - | </style>
| |
| - |
| |
| - | <script type="text/javascript">
| |
| - | $(document).ready(function(e) {
| |
| - | t = $('.fixed').offset().top;
| |
| - | mh = $('.main').height();
| |
| - | fh = $('.fixed').height();
| |
| - | $(window).scroll(function(e){
| |
| - | s = $(document).scrollTop();
| |
| - | if(s > t - 100){
| |
| - | $('.fixed').css('position','fixed');
| |
| - | if(s + fh - 6000 > mh){
| |
| - | $('.fixed').css('top',mh-s-fh+'px');
| |
| - | }
| |
| - | }else{
| |
| - | $('.fixed').css('position','');
| |
| - | }
| |
| - | })
| |
| - | });
| |
| - | </script>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="box">
| |
| - |
| |
| - | <div class="sub">
| |
| - | <div class="fixed">
| |
| - | <style type="text/css">
| |
| - | li{margin-bottom:0px;}
| |
| - | .cont ul,li{list-style: none;}
| |
| - | .cont ul {padding: 0; margin: 0;text-align:center;}
| |
| - | .cont .hmain{background-color:#0babe7 ;width: 220px;font-size:16px;float: left;border-top:#fff thin solid;border-bottom:#fff thin solid;}
| |
| - |
| |
| - | .cont a {text-decoration: none; /* padding-left: 10px;*/ display: block; display: inline-block; width: 220px;padding-top: 7px;padding-bottom: 7px;}
| |
| - | .cont .hmain a{color:#fff; }
| |
| - | .cont .hmain a:hover{color:#0babe7;background:#fff;border-top:#0babe7 thin solid;border-bottom:#0babe7 thin solid; }
| |
| - | .cont .hmain ul {display: none;}
| |
| - | .cont .hmain li a{font-size:14px;color:#fff;background-color:#8dc7e9;line-height:150%;border:#fff thin solid ;}
| |
| - | .cont .hmain li a:hover{color:#8dc7e9;background:#fff; line-height:150%;border:#8dc7e9 thin solid ;}
| |
| - | </style>
| |
| - |
| |
| - | <script type="text/javascript">
| |
| - | $(document).ready(function(){
| |
| - | $(".hmain").hover(function(){
| |
| - | $(this).children("ul").slideDown();
| |
| - | changeIcon($(this).children("a"));
| |
| - | },function(){
| |
| - | $(this).children("ul").slideUp();
| |
| - | changeIcon($(this).children("a"));
| |
| - | });
| |
| - | });
| |
| - | </script>
| |
| - | <div class="cont">
| |
| - | <ul>
| |
| - | <li class="hmain" style="margin-top:20px;">
| |
| - |
| |
| - | <li class="hmain">
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project">Overview</a>
| |
| - | </li>
| |
| - | <li class="hmain">
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Background">Background</a>
| |
| - | </li>
| |
| - | <li class="hmain">
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Design">Design</a>
| |
| - | </li>
| |
| - | <li class="hmain">
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/Endo-Induce">Experiment&Result</a>
| |
| - | <ul>
| |
| - |
| |
| - | <li>
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/Endo-Induce"><i> In vivo </i>Alkane Sensing Test</a>
| |
| - | </li>
| |
| - | <li>
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/Exo-Induce">Exogenous Alkanes Induce</a>
| |
| - | </li>
| |
| - | <li>
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/TetA">Endogenous Induce&Resistance Selection</a>
| |
| - | </li>
| |
| - | <li>
| |
| - | <a href="https://2013.igem.org/Team:Tianjin/Project/Experiment/LibraryConstruction">Library Construction</a>
| |
| - | </li>
| |
| - |
| |
| - | </ul>
| |
| - | </li>
| |
| - |
| |
| - | <li>
| |
| - | <a href="https://2013.igem.org/Main_Page" title="Main Page"><img src="https://static.igem.org/mediawiki/2013/7/75/IGEM-logo-blue.png" width="200px" height="200px" border="none" style="margin-top:80px;"/></a>
| |
| - | </li>
| |
| - | </ul>
| |
| - | </div>
| |
| - | </div>
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - | <div class="main">
| |
| - |
| |
| - | <center><span style="font-family:Arial;font-size:46px;color:#000;"> Experiment</span></center>
| |
| - | </html>
| |
| - |
| |
| - | = Exogenous Alkanes Induce=
| |
| - |
| |
| - |
| |
| - | <html>
| |
| - | <hr/>
| |
| - | <br/>
| |
| - | <p> Here, we want to test the response of Alk-Sensor to exogenous alkanes.</p>
| |
| - | <p>We add alkanes outside the cell to test the function of Alk-Sensor:</p>
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/0/0b/TJU-EX-1.png" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/0/0b/TJU-EX-1.png" width="500px" /></a></div>
| |
| - | <br/>
| |
| - |
| |
| - | <p><b> Construct:</b></p>
| |
| - | <p> Strain 1: Palkm and AlkR downstream of J23103, are cloned into two vectors--PSB3K3 and PSB1C3, respectively, which we hope to prevent the influence of the constitutive promoter J23103 on the reporter gene RFP.</p>
| |
| - | <p> Strain 2: We construct a blank vector without AlkR as blank control, and on the other vector the reporter gene RFP is still on the downstream of the inducible promoter PalkM.</p>
| |
| - | <p><b> Characterization: </b>The two strains are cultured in 3ml antibiotics （kan and cm） M9 medium for about 12 hours. Harvest the bacteria cells by centrifuge.</p>
| |
| - | <p><b> Result:</b></p>
| |
| - |
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/5/52/TJU-EX2.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/5/52/TJU-EX2.jpg" width="700px" /></a></div>
| |
| - | <br/>
| |
| - | <p> No. 55 is not red at all. No. 54 with water added shows a bit of red. No. 54 with alkanes added as inducers is the reddest among three. Alk-Sensor can respond to exogenous alkanes. Besides, No. 54 with water added is less red than with alkanes added because our sensor has a weaker response to water than to exogenous alkanes.</p>
| |
| - | <br/>
| |
| - | <h2>Specific detection </h2>
| |
| - |
| |
| - | <p><b> Characterization:</b> The two strains are cultured in M9 medium for about 12 hours. Adjust the concentration of suspension culture until OD reaches 0.6, then take out 3mL and add 300μL Octane(C8),Decane(C10),Dodecane(C12), Tetradecane(C14), Hexadecane(C16) and Octadecyl(C18) respectively. After that, induce for 24 hours. Measure the ratio of fluorescent intensity (fluorescent intensity per OD) of cells, which is shown below:</p>
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/7/7b/TJU-EX3.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/7/7b/TJU-EX3.jpg" width="700px" /></a></div>
| |
| - | <br/>
| |
| - |
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/7/78/Tju-ex1.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/7/78/Tju-ex1.jpg" width="700px" /></a></div>
| |
| - | <br/>
| |
| - |
| |
| - | <p> From this diagram, we can clearly see that induced by saturated Octane(C8), ratio of fluorescent intensity is the strongest. We infer that there are two causes of the low ratio of fluorescent intensity of other alkanes.</p>
| |
| - | <p>1.Alkanes are hydroforbic molecules, which makes alkanes with carbon chain lengths equal to or larger than 10 hard to dissolve in water to induce alkR. </p>
| |
| - | <p>2.Ratio of fluorescent intensity of Dodecane(C12) induced is larger than those induced by Decane(C10), proving that alkR has a specifical detection of Dodecane(C12). This is also consistent with the reference.</p>
| |
| - | <br/>
| |
| - | <h2>Upper limits of detection</h2>
| |
| - | <p>We choose Octane(C8), because it is commonly used to characterize alkane-sensor in most references, and we choose Pentadecane(C15), because our endogenous alkane producing module produce this kind of alkane. </p>
| |
| - |
| |
| - | <p>We choose the strains of No.54 and culture them in 3mL LB medium until OD reaches 0.6, add C8 and C15 of a concentration range from100nmol/L to 1mmol/L, over-night cultured. All of them are utilized to measure fluorescent intensity of cells.</p>
| |
| - |
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/b/bb/TJU-EX4.JPG" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/b/bb/TJU-EX4.JPG" width="700px" /></a></div>
| |
| - | <p>C8</p>
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/8/84/TJU-EX5.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/8/84/TJU-EX5.jpg" width="700px" /></a></div>
| |
| - | <p>C15</p>
| |
| - | <br/>
| |
| - | <P><b>Result:</b> With the increasing concentration of alkanes, ratio of fluorescent intensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, the increase of ratio of fluorescent intensity becomes smooth, approaching its upper limit at the concentration of 10μmol/L C8 in LB medium, and approaching its upper limit at the concentration of 1μmol/L C15 in LB medium.
| |
| - | </P>
| |
| - | <p> We choose the strains of No.54 and culture them in 3mL M9 medium until OD reaches 0.6,add C8 and C15 of a concentration range from 100nmol/L to 1mmol/L, over-night cultured. All of them are utilized to measure fluorescence intensity of cells.</p>
| |
| - |
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/7/7a/TJU-EX6.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/7/7a/TJU-EX6.jpg" width="700px" /></a></div>
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/a/a7/TJU-EX7.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/a/a7/TJU-EX7.jpg" width="700px" /></a></div>
| |
| - | <br/>
| |
| - | <p><b>Result:</b> With the increasing concentration of alkanes, ratio of fluorescent intensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, ratio of fluorescent intensity increases constantly. At the concentration of 10nmol/L to 2nmol/L of C8 and C15, stronger positive correlation is showed than higher concentration of C8 and C15.
| |
| - | </p>
| |
| - | <p> We choose the strains of No.54 and culture them in 3mL M9 medium, until OD reaches 0.6,add C8 and C15 of a concentration range from 100nmol/L to 1mmol/L and 30uL DMSO, over-night cultured. All of them are utilized to measure fluorescent intensity of cells.</p>
| |
| - |
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/a/ae/TJU-EX8.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/a/ae/TJU-EX8.jpg" width="700px" /></a></div>
| |
| - | <br/>
| |
| - | <div style="text-align:center;vertical-align:middle;"><a href="https://static.igem.org/mediawiki/2013/2/25/TJU-EX9.jpg" target="_blank" ><img src="https://static.igem.org/mediawiki/2013/2/25/TJU-EX9.jpg" width="700px" /></a></div>
| |
| - | <br/>
| |
| - | <p><b>Result:</b> With the increasing concentration of alkanes, ratio of fluorescent intensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, the increase of ratio of fluorescent intensity becomes smooth, approaching its upper limit at the concentration of 10μmol/L C8 in M9 medium, and approaching its upper limit at the concentration of 1μmol/L C15 in M9 medium. At the concentration of 10nmol/L to 2nmol/L of C8 and C15, strong positive correlation is also showed. </p>
| |
| - | <p>DMSO is a polar aprotic solvent which increases permeability of cell membranes and helps alkanes to induce alkR. However, compared with DMSO-free medium, growth of cells are inhibited.</p>
| |
| - |
| |
| - |
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div class="note2">
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - | <!--
| |
| - | <div class="note">
| |
| - |
| |
| - | </div>
| |
| - | <div class="note">
| |
| - |
| |
| - | </div>
| |
| - | -->
| |
| - |
| |
| - | </div>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <div id="footer">
| |
| - | <p><br />
| |
| - | <br />
| |
| - | E-mail：austinamens@gmail.com | Address:Building No.20, No.92 Weijin Road, Tianjin, China | Zip-code：300072
| |
| - | <br />Copyright 2013 © Tianjin University iGEM Team
| |
| - | </p>
| |
| - | </div>
| |
| - |
| |
| - | <!--top button section-->
| |
| - | <div style="display: block" id="goTopBtn">
| |
| - | <a href="#top" title="Top"><img border=0 src="https://static.igem.org/mediawiki/2013/6/61/Top.jpg"></a>
| |
| - | </div>
| |
| - |
| |
| - | </html>
| |
"
