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| - | <!-- *** What falls between these lines is the Alert Box! You can remove it from your pages once you have read and understood the alert *** -->
| + | {{Template:CU-BoulderHeader}} |
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| - | You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples <a href="https://2008.igem.org/Help:Template/Examples">HERE</a>.
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| - | You <strong>MUST</strong> have all of the pages listed in the menu below with the names specified. PLEASE keep all of your pages within your teams namespace.
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| - | {| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
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| - | !align="center"|[[Team:CU-Boulder|Home]]
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| - | !align="center"|[[Team:CU-Boulder/Team|Team]]
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| - | !align="center"|[https://igem.org/Team.cgi?year=2013&team_name=CU-Boulder Official Team Profile]
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| - | !align="center"|[[Team:CU-Boulder/Project|Project]]
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| - | !align="center"|[[Team:CU-Boulder/Parts|Parts Submitted to the Registry]]
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| - | !align="center"|[[Team:CU-Boulder/Modeling|Modeling]]
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| - | !align="center"|[[Team:CU-Boulder/Notebook|Notebook]]
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| - | !align="center"|[[Team:CU-Boulder/Safety|Safety]]
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| - | !align="center"|[[Team:CU-Boulder/Attributions|Attributions]]
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| - | |}
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| - | Restriction Enzymes are necessary tools in synthetic biology, without them, biobrick assembly would be impossible. These restriction enzymes are also very pricey and prove to be a significant cost in labs everywhere. Because these enzymes are expensive, but necessary, experiments in synthetic biology are limited to companies and universities with high budgets. Here at the University of Colorado-Boulder, we aim to find a technology that lowers the costs of these restriction enzymes and open the field of synthetic biology to more people.
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| - | Our solution to this problem we are trying to create is a "BetaBioBrick." Essentially we are trying to come up a with low cost method for labs to create their own restriction enzymes so they won't have to order from pricey suppliers. We attempting to do this by creating a BioBrick part that will house the genes for a restriction enzyme (EcoRI, XbaI, ApoI), their relative methylase, and if needed a method of purification.
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| - | <h1 class="firstHeading">Team:Colorado State/Project</h1>
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| - | ***************************************************************
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| - | *********STOP! BEFORE YOU STEAL MY CODE, READ THIS!************
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| - | ***************************************************************
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| - | ***************************************************************
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| - |
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| - | 2012 Colorado State iGEM Team Wiki Site
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| - |
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| - | by Ethan Ho - Ethan.N.Ho@Gmail.com
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| - |
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| - | All code is original, except the dropdown menu, which is modified
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| - | code.
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| - | Do not copy or in any way, shape, or form, use any of this code
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| - | without the explicit permission of Ethan Ho (reachable by above
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| - | E-mail). I'll probably say yes.
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| - | Be a good person. Obey the rules.
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| - | Peace out,
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| - | Ethan
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| - | text-align: center;
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| - | background: none;
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| - | margin: 0;
| |
| - | }
| |
| - |
| |
| - | #menu_bar dt {
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| - | text-align: center;
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| - | width: 100%;
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| - | padding: 8px;
| |
| - | font-weight: bold;
| |
| - | cursor: pointer;
| |
| - | background: #925F00; /*******COLOR*******/
| |
| - | }
| |
| - |
| |
| - | #menu_bar dd {
| |
| - | position: absolute;
| |
| - | overflow: hidden;
| |
| - | width: 267px;
| |
| - | display: none;
| |
| - | background: #E9B300; /*******COLOR*******/
| |
| - | z-index: 4;
| |
| - | opacity: 0;
| |
| - | }
| |
| - |
| |
| - | #menu_bar ul {
| |
| - | width: 263px;
| |
| - | border: 2px solid #925F00; /*******COLOR*******/
| |
| - | list-style: none;
| |
| - | border-top: none;
| |
| - | }
| |
| - |
| |
| - | #menu_bar li {
| |
| - | display: inline;
| |
| - | }
| |
| - |
| |
| - | #menu_bar a, #menu_bar a:active, #menu_bar a:visited {
| |
| - | display: block;
| |
| - | padding: 5px;
| |
| - | text-decoration: none;
| |
| - | width: 100%;
| |
| - | color: #925F00; /*******COLOR*******/
| |
| - | border-top: 1px solid #925F00; /*******COLOR*******/
| |
| - | }
| |
| - |
| |
| - | #menu_bar a:hover {
| |
| - | background: #F1C900; /*******COLOR*******/
| |
| - | }
| |
| - |
| |
| - | .leftBorder {
| |
| - | border-left: solid 2px #CCC;
| |
| - | }
| |
| - |
| |
| - | </style>
| |
| - | <script type="text/javascript">
| |
| - | //newOpac: sets new opacity
| |
| - |
| |
| - | //*******************************************************************
| |
| - | //************************SLIDESHOW SCRIPT***************************
| |
| - | //*******************************************************************
| |
| - |
| |
| - | //Performs fade animation; main function
| |
| - | function fade(curElemID,isFading) {
| |
| - |
| |
| - | //length that each slide shows before fade (in milliseconds)
| |
| - | var slideStayTime = 3000;
| |
| - |
| |
| - | //length of fade (milliseconds)
| |
| - | var fadeTime = 800;
| |
| - |
| |
| - | //number of fade loops: > means smoother, more CPU-intensive transition
| |
| - | var numFadeLoops = 8;
| |
| - |
| |
| - | //In order for fade to work correctly, opacity must be rounded to
| |
| - | //the appropriate number of decimal places. roundTo defines this.
| |
| - | //For numFadeLoops < 20, roundTo = 1 will suffice. roundNum is the
| |
| - | //functional form.
| |
| - | var roundTo = 1;
| |
| - | var roundNum = Math.pow(10, roundTo);
| |
| - |
| |
| - | //Fetches the current slide element
| |
| - | var curElem = document.getElementById('slide' + curElemID);
| |
| - |
| |
| - | //Performs pre-fade functions. Function must be called with isFading = false.
| |
| - | if (isFading == false) {
| |
| - |
| |
| - | //Fetches the next slide element. If there is no next slide,
| |
| - | //return to slide1.
| |
| - | var nextElem = document.getElementById('slide' + (curElemID + 1));
| |
| - | if (!nextElem) {
| |
| - | var nextElem = document.getElementById('slide1');
| |
| - | } //end if
| |
| - |
| |
| - | //Brings current slide to front/fading z-index (3) and puts the
| |
| - | //next slide in the corresponding z-index (2). Slides that are
| |
| - | //neither the current or next slide should have z-index = 1.
| |
| - | curElem.style.zIndex = 3;
| |
| - | nextElem.style.zIndex = 2;
| |
| - |
| |
| - | //Sets a preliminary opacities. Optional
| |
| - | curElem.style.opacity = 1;
| |
| - | curElem.style.filter = 'alpha(opacity=100)';
| |
| - | nextElem.style.opacity = 1;
| |
| - | nextElem.style.filter = 'alpha(opacity=100)';
| |
| - |
| |
| - | } //end if
| |
| - |
| |
| - | //Fades out the current slide if it is not already completely faded yet
| |
| - | if (parseFloat(curElem.style.opacity) > 0) {
| |
| - |
| |
| - | //Sets new opacity and reruns the function
| |
| - | var newOpac = (Math.round(roundNum * (parseFloat(curElem.style.opacity) - (1/numFadeLoops)))) / roundNum;
| |
| - | curElem.style.opacity = newOpac;
| |
| - | curElem.style.filter = 'alpha(opacity=' + (newOpac * 100) + ')';
| |
| - | setTimeout(function(){fade(curElemID)}, (fadeTime/numFadeLoops));
| |
| - |
| |
| - | //Current slide has faded completely
| |
| - | } else {
| |
| - |
| |
| - | //Sets the current slide to a hidden state with full opacity
| |
| - | curElem.style.zIndex = 1;
| |
| - | curElem.style.opacity = 1;
| |
| - | curElem.style.filter = 'alpha(opacity=100)';
| |
| - |
| |
| - | //Sets next slide element. If there is no next slide,
| |
| - | //return to slide1.
| |
| - |
| |
| - | nextElemID = getNextSlide(curElemID);
| |
| - |
| |
| - | //Starts next fade. isFading = false is a required parameter
| |
| - | setTimeout(function(){fade(nextElemID, false)}, slideStayTime);
| |
| - |
| |
| - | //Menu bar functions
| |
| - | menuBar(curElemID);
| |
| - |
| |
| - | } //end if
| |
| - |
| |
| - | } //end function fade
| |
| - |
| |
| - | //Finds the next slide and returns its 'slide' + x id.
| |
| - | function getNextSlide(curElemID) {
| |
| - |
| |
| - | var nextElem = document.getElementById('slide' + (curElemID + 1));
| |
| - | if (nextElem) {
| |
| - | var nextElemID = curElemID + 1;
| |
| - | } else {
| |
| - | var nextElemID = 1;
| |
| - | } //end if
| |
| - |
| |
| - | return nextElemID;
| |
| - |
| |
| - | } //end function getNextSlide
| |
| - |
| |
| - | //Changes menu_bar element in coordination with slides
| |
| - | function menuBar(curElemID) {
| |
| - |
| |
| - | nextElemID = getNextSlide(curElemID);
| |
| - | var curMenuElem = document.getElementById('ddHeader' + curElemID);
| |
| - | var nextMenuElem = document.getElementById('ddHeader' + nextElemID);
| |
| - |
| |
| - | //menu_bar item change actions go here
| |
| - | nextMenuElem.style.background = '#DC8800'; /*******COLOR*******/
| |
| - |
| |
| - | //Change last menu_bar item back to its normal state
| |
| - | curMenuElem.style.background = '#925F00'; /*******COLOR*******/
| |
| - |
| |
| - | } //end function menuBar
| |
| - |
| |
| - | //*******************************************************************
| |
| - | //************************DROPDOWN SCRIPT****************************
| |
| - | //*******************************************************************
| |
| - |
| |
| - | var ddspeed = 10;
| |
| - | var ddtimer = 15;
| |
| - |
| |
| - | //Main function to handle the mouse events
| |
| - | function ddMain(id, direction) {
| |
| - |
| |
| - | var headerElem = document.getElementById('ddHeader' + id);
| |
| - | var contentElem = document.getElementById('ddContent' + id);
| |
| - | clearInterval(contentElem.timer);
| |
| - |
| |
| - | if (direction == 1) {
| |
| - | clearTimeout(headerElem.timer);
| |
| - | if (contentElem.maxh <= contentElem.offsetHeight) {
| |
| - | if (contentElem.maxh) {
| |
| - | return;
| |
| - | } //end if
| |
| - | } else if (!contentElem.maxh) {
| |
| - | contentElem.style.display = 'block';
| |
| - | contentElem.style.height = 'auto';
| |
| - | contentElem.maxh = contentElem.offsetHeight;
| |
| - | contentElem.style.height = '0px';
| |
| - | } //end if
| |
| - | contentElem.timer = setInterval(function(){ddSlide(contentElem,1)},ddtimer);
| |
| - | } else {
| |
| - | headerElem.timer = setTimeout(function(){ddCollapse(contentElem)},50);
| |
| - | } //end if
| |
| - |
| |
| - | } //end function ddMain
| |
| - |
| |
| - | //Collapse menu
| |
| - | function ddCollapse(contentElem) {
| |
| - |
| |
| - | contentElem.timer = setInterval(function(){ddSlide(contentElem, -1)},ddtimer);
| |
| - |
| |
| - | } //end function ddCollapse
| |
| - |
| |
| - | //Cancel the collapse if a user rolls over the dropdown
| |
| - | function cancelCollapse(id) {
| |
| - |
| |
| - | var headerElem = document.getElementById('ddHeader' + id);
| |
| - | var contentElem = document.getElementById('ddContent' + id);
| |
| - | clearTimeout(headerElem.timer);
| |
| - | clearInterval(contentElem.timer);
| |
| - |
| |
| - | if (contentElem.offsetHeight < contentElem.maxh) {
| |
| - | contentElem.timer = setInterval(function(){ddSlide(contentElem,1)}, ddtimer);
| |
| - | } //end if
| |
| - |
| |
| - | }
| |
| - |
| |
| - | //Incrementally expand/contract the dropdown and change the opacity
| |
| - | function ddSlide(contentElem,direction) {
| |
| - |
| |
| - | var curHeight = contentElem.offsetHeight;
| |
| - |
| |
| - | if (direction == 1) {
| |
| - | distance = Math.round((contentElem.maxh - curHeight) / ddspeed);
| |
| - | } else {
| |
| - | distance = Math.round(curHeight / ddspeed);
| |
| - | } //end if
| |
| - |
| |
| - | if (distance <= 1) {
| |
| - | if (direction == 1) {
| |
| - | distance = 1;
| |
| - | } //end if
| |
| - | } //end if
| |
| - |
| |
| - | contentElem.style.height = curHeight + (distance * direction) + 'px';
| |
| - | contentElem.style.opacity = curHeight / contentElem.maxh;
| |
| - | contentElem.style.filter = 'alpha(opacity=' + (curHeight * 100 / contentElem.maxh) + ')';
| |
| - |
| |
| - | if (curHeight < 2) {
| |
| - | if (direction != 1) {
| |
| - | clearInterval(contentElem.timer);
| |
| - | } //end if
| |
| - | } else if (curHeight > (contentElem.maxh - 2)) {
| |
| - | if (direction == 1) {
| |
| - | clearInterval(contentElem.timer);
| |
| - | } //end if
| |
| - | } //end if
| |
| - | } //end function ddSlide
| |
| - |
| |
| - | //*******************************************************************
| |
| - | //************************MISC FUNCTIONS*****************************
| |
| - | //*******************************************************************
| |
| - |
| |
| - | //Javascript link function
| |
| - | function linkTo(url) {
| |
| - |
| |
| - | window.location.href = url;
| |
| - |
| |
| - | } //end function linkTo
| |
| - |
| |
| - | //Simple function that performs startup scripts when prompted
| |
| - | function startFunctions () {
| |
| - |
| |
| - | setTimeout('fade(1,false)', 2000);
| |
| - | menuBar(0);
| |
| - |
| |
| - | } //end function startFunctions
| |
| - |
| |
| - | </script>
| |
| - | </head>
| |
| - | <body>
| |
| - | <img src="https://lh6.googleusercontent.com/-1Q-9hddFWuI/UCGVfn6W4wI/AAAAAAAAAVQ/e7PcCnXWO8o/s500/igem_home_logo.png" width="100" height="100" style="position: fixed; top: 10px; right: 10px; cursor: pointer;" onClick="linkTo('https://2012.igem.org/Main_Page')" />
| |
| - | <div style="border: solid red 1px;" id="menubar"></div>
| |
| - | <a id="side_bar" href="https://2012.igem.org/Team:Colorado_State/Safety"></a>
| |
| - | <div id="content_top">
| |
| - | <div id="slideshow">
| |
| - | <div style="position: absolute; top: 0; left: 0; height: 100%; width: 55%; z-index: 4; background: url(https://lh4.googleusercontent.com/-DDU8elUPsNM/UAm_sNRqnMI/AAAAAAAAARE/1Z2Xnvq4J1A/s600/fade1.png) top left; background-size: 100% 100%;">
| |
| - | <a href="https://2012.igem.org/Team:Colorado_State">
| |
| - | <div style="position: relative; top: 30px; left: 10px; border: solid red 1px; height: 220px; width: 287.65px; border: none; background: url(https://lh3.googleusercontent.com/-31sRsyZb5LU/UCKUYpC-gMI/AAAAAAAAAVk/MiL9X1XsNQ0/s640/logo2.png) no-repeat left; background-size: auto 100%;">
| |
| - | <img src="http://picasion.com/pic56/66eccba80910fafd431232af43e6e48f.gif" style="position: absolute; top: 12%; left: 28%; height: 22%; width: auto;" />
| |
| - | <img src="http://picasion.com/pic56/2f058d1a44eeb5749a3117dd586e79d6.gif" style="position: absolute; top: 26%; left: 21%; height: 12%; width: auto;" />
| |
| - | <img src="http://picasion.com/pic56/fc61f0752651a6be3920ad3917e72fa2.gif" style="position: absolute; top: 42%; left: 1.5%; height: 12%; width: auto;" />
| |
| - | </div>
| |
| - | </a>
| |
| - | </div>
| |
| - | <img id="slide1" class="slide" src="https://lh6.googleusercontent.com/-HUO1pRHwGn8/UA6_152AUUI/AAAAAAAAAUg/NIAPTu0rJeo/s912/slide2.jpg" style="z-index: 3;" />
| |
| - | <img id="slide2" class="slide" src="https://lh4.googleusercontent.com/-wv_cz741rxA/UA6_2SesX8I/AAAAAAAAAUo/kIBZr3Ydpi4/s912/slide1.jpg" />
| |
| - | <img id="slide3" class="slide" src="https://lh4.googleusercontent.com/-Atg7LS0jvu8/UA7Br49KLMI/AAAAAAAAAU8/LyqAernMIrM/s600/Brewery2-large.jpeg" />
| |
| - | </div>
| |
| - | <table id="menu_bar" cellspacing="0">
| |
| - | <tr>
| |
| - | <td>
| |
| - | <dl>
| |
| - | <dt id="ddHeader1" onClick="linkTo('https://2012.igem.org/Team:Colorado_State')">Home</dt>
| |
| - | <dd id="ddContent1">
| |
| - | <ul>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Project">Home</a></li>
| |
| - | </ul>
| |
| - | </dd>
| |
| - | </dl>
| |
| - | </td>
| |
| - | <td>
| |
| - | <dl>
| |
| - | <dt id="ddHeader2" class="leftBorder" onMouseOver="ddMain(2,1)" onMouseOut="ddMain(2)">The Brew</dt>
| |
| - | <dd id="ddContent2" onMouseOver="cancelCollapse(2)" onMouseOut="ddMain(2)">
| |
| - | <ul>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Project">Project Summary</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Parts">Parts Submitted to the Registry</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Results">Results</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Safety">Safety</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Modeling">Modeling</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Notebook">Team Notebook</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Calendar">Calendar</a></li>
| |
| - | </ul>
| |
| - | </dd>
| |
| - | </dl>
| |
| - | </td>
| |
| - | <td>
| |
| - | <dl>
| |
| - | <dt id="ddHeader3" class="leftBorder" onMouseOver="ddMain(3,1)" onMouseOut="ddMain(3)">The Brewers</dt>
| |
| - | <dd id="ddContent3" onMouseOver="cancelCollapse(3)" onMouseOut="ddMain(3)">
| |
| - | <ul>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Team">Meet the Students</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Instructors">Meet the Instructors</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Advisors">Meet the Advisors</a></li>
| |
| - | <li><a href="https://igem.org/Team.cgi?year=2012&team_name=Colorado_State">Official Team Page</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Sponsors">Sponsors</a></li>
| |
| - | <li><a href="https://2012.igem.org/Team:Colorado_State/Attributions">Attributions</a></li>
| |
| - | </ul>
| |
| - | </dd>
| |
| - | </dl>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </table>
| |
| - | </div>
| |
| - | <script type="text/javascript">startFunctions();</script>
| |
| - | <div id="content_main">
| |
| - |
| |
| - |
| |
| - |
| |
| - | <!--START PAGE CONTENT-->
| |
| - |
| |
| - | <a class="editbutton" href="https://2012.igem.org/wiki/index.php?title=Team:Colorado_State/Project&action=edit">Edit page</a>
| |
| - |
| |
| - | <h1>Project Summary</h1>
| |
| - | <p>We are currently pursuing the concept of brewing a gluten-free beer using a yeast that secretes an enzyme to break down gluten.</p>
| |
| - | <br />
| |
| - | <p>As Fort Collins is a major brewing hub, it was natural for our team to gravitate toward a beer-related project. Knowing full well the problems caused by Celiac disease, and the affinity many others have for reducing gluten in their diets, we decided to design and create a yeast strain capable of both fermenting quality beer, and breaking down gluten. To accomplish this we had to address several issues:</p>
| |
| - |
| |
| - | <h3>What is the immunological basis for gluten intolerance i.e. gluten’s antigenic qualities?</h3>
| |
| - |
| |
| - | <p>Celiac disease is an autoimmune disorder that affects the digestive system. People with Celiac disease suffer from a severe reaction when exposed to gluten, a protein found in wheat, rye, and barley. Glutamine and proline rich regions of gluten family proteins, which include gliadin in wheat and hordein in barley, are incredibly stable and cannot be broken down by the stomach. The antigenicity of gluten seems to be due to these proline and glutamine rich peptides, the most prevalent one being the 33-mer: LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, found in gliadin. The sequence seems to be fairly conserved across barley and wheat. Three distinct patient-specific T cell epitopes identified previously in T cell proliferation assays are present in this peptide, namely, PFPQPQLPY, PQPQLPYPQ (three copies), and PYPQPQLPY (two copies). Upon reaching the intestines, these peptides are processed by a Celiac’s immune system, producing a response that damages villi in the small intestine and interferes with absorption of nutrients from food.<sup><a href="http://digestive.niddk.nih.gov/ddiseases/pubs/celiac/">[1]</a> <a href="http://www.sciencemag.org/content/297/5590/2275.full">[2]</a></sup></p>
| |
| - |
| |
| - | <h3>Where can we find a viable enzyme?</h3>
| |
| - |
| |
| - | <p>Our search for an enzyme capable of breaking down gluten and neutralizing its toxicity led us first to the enzyme mutated by the 2011 UW iGEM team. The modified Kumamolisin-As has a maximal activity at a pH of 4 and would work well in the pH range of 5.2-5.5 found in beer. For expression in yeast we had to account for codon bias, and optimized the sequence so it could more easily be moved from a prokaryotic system to a eukaryotic one. Another promising enzyme was AN-PEP. This protease already cleaves after proline residues and is stable at low pH. Used in the cocktail known as Brewers Clarex, AN-PEP has been shown to reduce gluten levels in beer when it is added to a final product. Unfortunately, the enzyme is protected by patent and was unavailable to us. </p>
| |
| - |
| |
| - | <h3>How will the enzyme be secreted?</h3>
| |
| - | <p>Yeast have two mating types, called “a” and “<math>$\alpha$</math>”. Yeast of the alpha mating type secrete mating factor-alpha (MF-<math>\alpha</math>), which is tagged with a secretion factor at the N-terminus of the protein. Experimental evidence shows that this signal sequence is cleaved in the golgi before MF-<math>\alpha</math> is exported from the cell. It is commonly used by researchers to mark foreign proteins for secretion in laboratory yeast. The tag’s sequence was placed directly upstream of the sequence for Kumamolisin. The DNA itself was synthesized by IDT, but we were also able to extract it from the yeast genome by PCR.<sup><a href="http://www.jbc.org/content/263/13/6209.full.pdf">[1]</a> <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC391546/pdf/pnas00616-0033.pdf">[2]</a></sup></p>
| |
| - |
| |
| - | <h3>How will we move our system into yeast?</h3>
| |
| - |
| |
| - |
| |
| - | <p>Yeast can take up plasmids from the environment, but without selective pressure will drop them quickly. This would not be ideal for our purposes, as it would be unrealistic and potentially harmful to add antibiotics to every batch of beer just to ensure plasmid retention. As a proof of concept we introduced our system into yeast using plasmids, but we were also in the process of developing an integrative plasmid that would insert our sequence directly into the yeast genome. </p>
| |
| - | <br />
| |
| - |
| |
| - | <p>Two plasmids were used:</p>
| |
| - | <ul>
| |
| - |
| |
| - | <li>pCM189: centromeric yeast plasmid, marker URA3, tetracycline repressed expression of target gene under control of tetO2, low copy number</li>
| |
| - |
| |
| - | <li>pCM190: episomal yeast plasmid, marker URA3, tetracycline repressed expression of target gene under control of tetO7, high copy number</li>
| |
| - |
| |
| - | <li> The integrative plasmid was constructed by adding the resistance gene for geneticin to pCM189/MF-alpha/Kuma-max.</li>
| |
| - | </ul>
| |
| - |
| |
| - |
| |
| - | <h3>How will we assay the enzyme and screen for gluten?</h3>
| |
| - | <p>Before we move our engineered yeast into beer, it will be necessary to determine whether or not the enzyme is performing as expected. In 2011 the UW iGEM team assayed mutated Kumamolisin-As using fluorescence. We hope to repeat their tests in the lab using the peptide sequence PQPQLP with attached fluorophore and quencher. When the sequence is cleaved, the fluorophore will be separated from the quencher and will fluoresce. Ideally our results will resemble Washington’s.</p>
| |
| - | <br />
| |
| - | <p>Once we have determined whether the enzyme is present, and how active it is, the next step will be to assess how well it can break down the naturally occurring peptides found in beer. Studies have shown that the brewing process breaks native gluten proteins into smaller peptides, some of which are still immunogenic. However, it will still be necessary to confirm that Kumamolisin-As can manage these peptides and neutralize their toxicity. To do so, we have prepared a competitive ELISA to detect the presence of gluten antigens after incubation with Kumamolisin-As.</p>
| |
| - |
| |
| - | <h3>What will be the effects of the secreted protein on beer?</h3>
| |
| - |
| |
| - | <p>Flavor/head/haze</p>
| |
| - |
| |
| - | <h1>Project Details</h1>
| |
| | | | |
| | + | <div class="shadow-bottom"> |
| | + | <dl> |
| | + | <dt>The 2013 CU-Boulder iGEM Project</dt> |
| | + | <p></p> |
| | + | <p>The main focus of the iGEM project here at CU-Boulder is to help make synthetic biology more accessible and affordable. We spent the summer developing parts, procedures, and documentation to help make this vision a reality. The original goal was to create the constructs and purification methods necessary to produce and isolate restriction enzymes. Along the way we explored some novel approaches to DNA and protein purification and developed experimentally tested protocols for these and other procedures essential to Biobrick assembly. Our purification methods exemplify the ideal of using common lab materials to make performing everyday lab techniques as accessible and inexpensive as possible. A related aspect of our project was exploring methods of recycling consumables associated with lab work in order to reduce waste and material expenses. We hope that our findings using this "do-it-yourself" approach of synthetic biology help make this type of research more accessible for those where funding is a limiting factor. |
| | + | </p> |
| | <ul> | | <ul> |
| - | <li>Yeast
| + | <li><a href="https://2013.igem.org/Team:CU-Boulder/Project/Kit/RestrictionEnzymes">Restriction Enzymes</a></li> |
| - | <ul>
| + | <li><a href="https://2013.igem.org/Team:CU-Boulder/Project/Kit/Purification">Protein Purification</a></li> |
| - | <li>Assemble plasmids
| + | <li><a href="https://2013.igem.org/Team:CU-Boulder/Project/Kit/DNAPurification">DNA Purification</a></li> |
| - | <ul>
| + | |
| - | <li>Integrative: industrial strain</li>
| + | |
| - | <li>Centromeric: Ethan</li>
| + | |
| - | <li>Episomal: Guy</li>
| + | |
| - | </ul>
| + | |
| - | </li>
| + | |
| - | <li>His Tag Plasmids: Dave
| + | |
| - | <ul>
| + | |
| - | <li>C-terminus</li>
| + | |
| - | <li>N-terminus</li>
| + | |
| - | </ul>
| + | |
| - | </li>
| + | |
| - | <li>Clone mating factor alpha: Steven</li>
| + | |
| - | </ul>
| + | |
| - | </li>
| + | |
| - | <li>Bacteria
| + | |
| - | <ul>
| + | |
| - | <li>Assemble plasmids (3A): Steven</li>
| + | |
| - | <li>His Tag Plasmids: Dave
| + | |
| - | <ul>
| + | |
| - | <li>C-terminus</li>
| + | |
| - | <li>N-terminus</li>
| + | |
| - | </ul>
| + | |
| - | </li>
| + | |
| - | <li>Secretion: Ethan</li>
| + | |
| - | </ul>
| + | |
| - | </li>
| + | |
| - | <li>Functional Protease Assay
| + | |
| - | <ul>
| + | |
| - | <li>Flourophore/Quencher: Ryan</li>
| + | |
| - | <li>Mass Spec: Ryan/Guy</li>
| + | |
| - | <li>Elisa: Guy</li>
| + | |
| - | </ul>
| + | |
| - | </li>
| + | |
| - | <li>Modeling: Dave/Ryan/Ethan</li>
| + | |
| | </ul> | | </ul> |
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