Difference between revisions of "Team:Cadets2Vets/Design"

Cadets2Vets utilized literature searches and the iGEM Registry of Standard Biological Parts to identify the appropriate parts and sequences to use for the arsenic circuit. We knew we needed to have unregulated expression of ArsR and that ArsR needed to control expression of the GFP reporter. We found constitutive and regulated promoters to fit our needs.

Then we used DNA sequence analysis software, like Genome Compiler, to assemble a virtual sequence of what we needed. We used this software to add in additional features like the BioBrick Prefix and Suffix, as well as make predictions regarding the cloning steps we would take to create the arsenic circuit. The sequences of the Parts we selected can be found here.

To test the plasmids, we would use a variety of assays that would assess whether the correct proteins were being generated and whether the proteins were functioning as expected.

The creation of the circuit was achieved by obtaining synthesized DNA from Integrated DNA Technologies and using enzymes and competent E. coli cells obtained from New England Biolabs’ BioBrick Assembly Kit. DNA was cut using restriction enzymes and then linked to a vector backbone or to another piece of DNA using T4 ligase. The plasmid was then transformed into bacteria and then the plasmid DNA was isolated. We screened for the correct plasmid by using restriction enzyme digest and seeing that the resulting fragment matched our predictions.

In general, the strategy was to:

determine that proteins of the correct size were being produced
determine that the GFP was fluorescing when we expected it to fluoresce
optimize the assay conditions for the in vitro transcription/translation protocol
optimize transfer of the assay to paper tickets.

Our initial assessments of the plasmids were performed by monitoring the effect of a range of arsenic concentrations on the arsenic circuit being expressed by live E. coli cells, examining GFP fluorescence using a Light Cycler as a spectrophotometer, and looking at protein expression using a protein gel.

As we move forward in testing our circuit, we hope to identify the concentration range of arsenic that our circuit can report; the working conditions that the circuit will function, such as temperature and incubation time of the assay; and the specificity of the circuit. Additionally, our team is developing a portable imaging system using technology made by ECBC.

The use of cell-free extracts is important to the progress and future development of the arsenic circuit, and other similarly styled circuits.

The first advantage is that the use of cell-free extracts increases the shelf-life of the assay. We expect to be able to lyophilize the enzymes and proteins used in the arsenic circuit, so we do not have to be concerned with keeping bacteria cells alive and healthy.

The second advantage is that there will be no chance of accidentally releasing a modified bacteria to the environment. Although there is a low risk that the bacteria we use would adversely affect the environment, due to the required selective pressure needed to maintain the plasmid and the relative safety of the produced proteins, we will maintain laboratory safety standards and laws that prohibit the release of genetically modified organisms.

The third advantage is that we expect to be able to detect a broader range of arsenic using a cell-free system. Using live cells requires arsenic to be able to transport into the cell and interact with the necessary proteins. Having the proteins readily available in solution should reduce the barrier of arsenic accessing ArsR.

Difference between revisions of "Team:Cadets2Vets/Design"

Latest revision as of 02:49, 1 November 2017

DESIGN

Our circuit utilizes an arsenic regulatory protein (Arsr) and the green fluorescent protein (GFP) gene so that when Plasmid-containing cells are exposed to arsenic ions, gfp is produced, making the ticket glow under a blue or ultraviolet light.

the Thought Process

design

build

test

cell-free lysate

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2304 Jefferson Ave, Tacoma, WA 98402

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+			<a class="removable-parent" href="safety.html" data-link-type="EXISTING"   target="_self" >
-			<span id="vbid-59a5b-c9f8qbif"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Design</span>
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+			<a class="removable-parent" href="team.html" data-link-type="EXISTING"   target="_self" >
-			<span id="vbid-59a5b-cxv3wrcd"  class="preview-element Link item-link magic-circle-holder text-element custom" data-menu-name="PREVIEW_LINK" >Demonstrate</span>
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@@ Line 379: / Line 492: @@
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+	<h1 id="vbid-59a5b-dnp8mtfr" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >Our circuit utilizes an arsenic regulatory protein (Arsr) and the green fluorescent protein (GFP) gene so that when Plasmid-containing cells are exposed to arsenic ions, gfp is produced, making the ticket glow under a blue or ultraviolet light. <br></h1>
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-		<p>THE FOUNDATION OF OUR CIRCUIT</p>
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 </div>
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-		<ul style="box-sizing: border-box; line-height: 1.5em; list-style-type: square; margin: 0.3em 0px 0px 1.6em; padding-top: 0px; padding-right: 20px; padding-bottom: 0px; list-style-image: url(&quot;https://2017.igem.org/wiki/skins/Igem/resources/bullet.gif?2015-09-09T00:46:40Z&quot;); font-size: 13px; font-family: Tahoma, Geneva, sans-serif; color: rgb(0, 0, 0); text-align: start;"><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Test the efficacy of the plasma circuit in S.I.T.U.   </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Perform above experiments with prepared plasma circuit   </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Titrate both circuit and trigger to find optimal mixture   </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Standardize reactions    </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Test on paper matrix</font></span></li></ul>
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 							<div class="preview-content-holder shrinker-content">
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-<div class="element-placeholder" data-elementtype='TITLE' style="display:none;"></div>
 <br class="upper-line-break" />
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+<div class="preview-title-holder removable-parent order-handle">
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+	<div id="vbid-88f5fc9c-t14shpss" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
-		<ul style="box-sizing: border-box; line-height: 1.5em; list-style-type: square; margin: 0.3em 0px 0px 1.6em; padding-top: 0px; padding-right: 20px; padding-bottom: 0px; list-style-image: url(&quot;https://2017.igem.org/wiki/skins/Igem/resources/bullet.gif?2015-09-09T00:46:40Z&quot;); font-size: 13px; font-family: Tahoma, Geneva, sans-serif; color: rgb(0, 0, 0); text-align: start;"><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFF0;"><font style="box-sizing: border-box; font-size: 18px;">Test efficacy of freeze drying circuit on paper for distribution and storage purposes.  </font>  </span></li></ul>
+		<p><span id="docs-internal-guid-1b7da29c-f973-ba3d-a6ea-b5f9a7774632"><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">Cadets2Vets utilized literature searches and the <a data-cke-saved-href="http://parts.igem.org/Catalog" href="http://parts.igem.org/Catalog">iGEM Registry of Standard Biologic</a><span style="color:#000000;"><a data-cke-saved-href="http://parts.igem.org/Catalog" href="http://parts.igem.org/Catalog">al Parts</a> to identify the appropriate parts and sequences to use for the arsenic circuit. We knew we needed to have unregulated expression of ArsR and that ArsR needed to control expression of the GFP reporter. We found constitutive and regulated promoters to fit our needs.</span></span></span></p><p><br></p><p><span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><span style="color:#000000;">Then we used DNA sequence analysis software, like Genome Compiler, to assemble a virtual sequence of what we needed. We used this software to add in additional features like the BioBrick Prefix and Suffix, as well as make predictions regarding the cloning steps we would take to create the arsenic circuit. The sequences of the Parts we selected can be found </span><a data-cke-saved-href="https://2017.igem.org/Team:Cadets2Vets/Parts" href="https://2017.igem.org/Team:Cadets2Vets/Parts"><span style="color:#0000CD;">here</span></a><span style="color:#000000;">.</span></span></span></p><p><br></p><p><span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><span style="color:#000000;">To test the plasmids, we would use a variety of assays that would assess whether the correct proteins were being generated and whether the proteins were functioning as expected.</span></span></span></p>
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+	<h2 id="vbid-34ff8b6b-wmxuvsaa" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >build</h2>
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+	<div id="vbid-34ff8b6b-rhpe0gbn" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
+		<p><span id="docs-internal-guid-1b7da29c-f973-ba3d-a6ea-b5f9a7774632"><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"></span></span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"></span><span style="letter-spacing: 1px; background-color: transparent; font-size: 12pt; vertical-align: baseline; white-space: pre-wrap;">The creation of the circuit was achieved by obtaining synthesized DNA from Integrated DNA Technologies and using enzymes and competent </span><span style="letter-spacing: 1px; background-color: transparent; font-size: 12pt; font-style: italic; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="letter-spacing: 1px; background-color: transparent; font-size: 12pt; vertical-align: baseline; white-space: pre-wrap;"> cells obtained from New England Biolabs’ BioBrick Assembly Kit. DNA was cut using restriction enzymes and then linked to a vector backbone or to another piece of DNA using T4 ligase. The plasmid was then transformed into bacteria and then the plasmid DNA was isolated. We screened for the correct plasmid by using restriction enzyme digest and seeing that the resulting fragment matched our predictions. </span><span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><span style="color:#000000;"></span></span></span><br></p>
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-<div class="preview-divider-holder removable-parent order-handle">
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+		<p><span style="font-size:16px;">In general, the strategy was to:</span></p><ol><li><span style="font-size:16px;">determine that proteins of the correct size were being produced</span></li><li><span style="font-size:16px;">determine that the GFP was fluorescing when we expected it to fluoresce</span></li><li><span style="font-size:16px;">optimize the assay conditions for the in vitro transcription/translation protocol</span></li><li><span style="font-size:16px;">optimize transfer of the assay to paper tickets. </span></li></ol><p dir="ltr"><span style="font-size:16px;">Our initial assessments of the plasmids were performed by monitoring the effect of a range of arsenic concentrations on the arsenic circuit being expressed by live E. coli cells, examining GFP fluorescence using a Light Cycler as a spectrophotometer, and looking at protein expression using a protein gel.</span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">As we move forward in testing our circuit, we hope to identify the concentration range of arsenic that our circuit can report; the working conditions that the circuit will function, such as temperature and incubation time of the assay; and the specificity of the circuit. Additionally, our team is developing a portable imaging system using technology made by ECBC.</span></p>
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-	<h2 id="vbid-6443b981-zemrqugm" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >Testing our Product<br></h2>
+	<h2 id="vbid-bd0b19d9-fcd5o48r" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >cell-free lysate</h2>
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-<div class="preview-divider-holder removable-parent order-handle">
+<div class="preview-body-holder removable-parent order-handle">
-	<div id="vbid-6443b981-czwbea7x" class="preview-element preview-divider magic-circle-holder quick-text-style-menu" data-menu-name="PREVIEW_DIVIDER">
+	<div id="vbid-bd0b19d9-x6fcw58a" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
+		<p><span id="docs-internal-guid-1b7da29c-f973-ba3d-a6ea-b5f9a7774632"><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"></span></span><span style="font-size:16px;">The use of cell-free extracts is important to the progress and future development of the arsenic circuit, and other similarly styled circuits.</span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">The first advantage is that the use of cell-free extracts increases the shelf-life of the assay. We expect to be able to lyophilize the enzymes and proteins used in the arsenic circuit, so we do not have to be concerned with keeping bacteria cells alive and healthy. </span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">The second advantage is that there will be no chance of accidentally releasing a modified bacteria to the environment. Although there is a low risk that the bacteria we use would adversely affect the environment, due to the required selective pressure needed to maintain the plasmid and the relative safety of the produced proteins, we will maintain laboratory safety standards and laws that prohibit the release of genetically modified organisms.</span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">The third advantage is that we expect to be able to detect a broader range of arsenic using a cell-free system. Using live cells requires arsenic to be able to transport into the cell and interact with the necessary proteins. Having the proteins readily available in solution should reduce the barrier of arsenic accessing ArsR. </span></p>
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-	<div class="login dialog-holder">
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-		<form id="login-form">
-			<img id="user-gravatar" src="https://www.gravatar.com/avatar/?d=mm" /><br/>
-			<input name="name" type="text" placeholder="nickname/email" data-placeholder="nickname/email" /><br/>
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-			<input id="login-btn" class="clickable" type="button" class="input-btn" value="LOGIN">
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-			<div id="goto-login" class="clickable t-t">Back</div>
-		</form>
-	</div>
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-	</div>
-	<div id="fade" class="black_overlay fadein-noscript" style="display:none;"></div>
-</html>
-<html>
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-	// This is the jquery part of your template.
-	// Try not modify any of this code too much since it makes your menu work.
-	$(document).ready(function() {
-		$("#HQ_page").attr('id','');
-		// call the functions that control the menu
-		menu_functionality();
-		hide_show_menu();
-		//this function controls the expand and collapse buttons of the menu and changes the +/- symbols
-		function menu_functionality() {
-			//when clicking on a "menu_button", it will change the "+/-" accordingly and it will show/hide the corresponding submenu
-			$(".menu_button").click(function(){
-				// add or remove the class "open" , this class holds the "-"
-				$(this).children().toggleClass("open");
-				// show or hide the submenu
-				$(this).next('.submenu_wrapper').fadeToggle(400);
-			});
-			// when the screen size is smaller than 800px, the display_menu_control button appears and will show/hide the whole menu
-			$("#display_menu_control").click(function(){
-				$('#menu_content').fadeToggle(400);
-			});
-			// call the current page highlight function
-			highlight_current_page();
-		}
-		// call the highlight current page function to show it on the menu with a different background color
-		function highlight_current_page() {
-			// select a page from the menu based on the id assigned to it and the current page name and add the class "current page" to make it change background color
-			$("#"+  wgPageName.substring(wgPageName.lastIndexOf("/")+1, wgPageName.length ) + "_page").addClass("current_page");
-			// now that the current_page class has been added to a menu item, make the submenu fade in
-			$(".current_page").parents(".submenu_wrapper").fadeIn(400);
-			// change the +/- symbol of the corresponding menu button
-			$(".current_page").parents(".submenu_wrapper").prev().children().toggleClass("open");
-		}
-		// allow button on the black menu bar to show/hide the side menu
-		function hide_show_menu() {
-			// in case you preview mode is selected, the menu is hidden for better visibility
-			if (window.location.href.indexOf("submit") >= 0) {
-				$(".igem_2017_menu_wrapper").hide();
-			}
-			// if the black menu bar has been loaded
-  			if (document.getElementById('bars_item')) {
-				// when the "bars_item" has been clicked
-				$("#bars_item").click(function() {
-					$("#sideMenu").hide();
-					// show/hide the menu wrapper
-					$(".igem_2017_menu_wrapper").fadeToggle("100");
-				});
-  			}
-			// because the black menu bars loads at a different time than the rest of the page, this function is set on a time out so it can run again in case it has not been loaded yet
-			else {
-    				setTimeout(hide_show_menu, 15);
-			}
-		}
-	});
-</script>
-<style>
-/***************************************************** DEFAULT WIKI SETTINGS  ****************************************************/
-	/* Clear the default wiki settings */
-	#home_logo, #sideMenu { display:none; }
-	#sideMenu, #top_title, .patrollink  {display:none;}
-	#content { width:100%; padding:0px;  margin-top:-7px; margin-left:0px;}
-	body {background-color:white; }
-	#bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px; }
-/**************************************************************** MENU ***************************************************************/
-	/* Wrapper for the menu */
-	.igem_2017_menu_wrapper {
-		width: 15%;
-		height:100vh;
-		position:fixed;
-		right:0%;
-		padding:0px;
-		float:right;
-		border-left: 1px solid #383838;
-		background-color:#7c6429;
-		text-align:left;
-		font-family:Tahoma, Geneva, sans-serif;
-		overflow-y: auto;
-		overflow-x: hidden;
-	}
-	/* this hides the scrollbar to keep view consistency */
-	.igem_2017_menu_wrappe::-webkit-scrollbar {
-		display: none;
-	}
-	/* styling for links in the menu, removes the line under text */
-	.igem_2017_menu_wrapper a {
-		text-decoration: none;
-	}
-	/* styling for the images in the menu */
-	.igem_2017_menu_wrapper img {
-		width: 100%;
-	}
-	/* styling for the menu buttons */
-	.igem_2017_menu_wrapper .menu_button {
-		width: 100%;
-		padding: 10px 0px 10px 15px;
-		float:left;
-		border-bottom: 1px solid #383838;
-		font-size: 12px;
-		font-weight: bold;
-		color: #ffffff;
-		cursor: pointer;
-	}
-	.igem_2017_menu_wrapper .menu_bottom_padding {
-		width: 100%;
-		height: 30px;
-		float:left;
-	}
-	.menu_button.direct_to_page {
-		padding-left: 36px;
-	}
-	.igem_2017_menu_wrapper .menu_button .expand_collapse_icon {
-		width:10%;
-		float:left;
-	}
-	.igem_2017_menu_wrapper .menu_button .expand_collapse_icon::before {
-		content: "+";
-	}
-	.open::before {
-		content: "-" !important;
-	}
-	/* styling for the menu buttons on hover */
-	.igem_2017_menu_wrapper .menu_button:hover, .igem_2017_menu_wrapper .submenu_button:hover ,  .submenu_button.current_page:hover {
-		background-color: #3399ff;
-		text-decoration: none;
-		color:#ffffff;
-	}
-	/* styling for the menu button when it is the current page */
-	 .current_page {
-		background-color:#7fc1f7  !important;
-		color:#ffffff !important;
-	}
-	/* styling for the submenu buttons */
-	.igem_2017_menu_wrapper .submenu_button {
-		width: 100%;
-		padding: 10px 0px 10px 34px;
-		float:left;
-		background-color:#7c6429;
-		border-bottom: 1px solid #383838;
-		font-size: 12px;
-		color: #ffffff;
-		cursor: pointer;
-	}
-	/* wrapper for the submenu items, they are hidden by default*/
-	.igem_2017_menu_wrapper .submenu_wrapper {
-		display:none;
-	}
-	/* when the page size is bigger than 800px, this show/hide control is hidden by default */
-	.igem_2017_menu_wrapper #display_menu_control {
-		display:none;
-		text-align:center;
-	}
-/***************************************************** CONTENT OF THE PAGE ****************************************************/
-	/* Wrapper for the content */
-	.igem_2017_content_wrapper {
-		width: 81%;
-		margin: 2%;
-		display:block;
-		float:left;
-		background-color:white;
-		font-family:Tahoma, Geneva, sans-serif;
-	}
-/********************************* HTML STYLING  *********************************/
-	/* styling for the titles h1 h2 */
-	.igem_2017_content_wrapper h1, .igem_2017_content_wrapper h2 {
-		padding:5px 15px;
-		border-bottom: 0px;
-		color: #3399ff;
-	}
-	/* styling for the titles  h3 h4 h5 h6*/
-	.igem_2017_content_wrapper h3, .igem_2017_content_wrapper h4, .igem_2017_content_wrapper h5, .igem_2017_content_wrapper h6 {
-		padding:5px 15px;
-		border-bottom:0px;
-		color: #000000;
-	}
-	/* font and text */
-	.igem_2017_content_wrapper p {
-		padding: 0px 15px;
-		font-size: 13px;
-	}
-	/* Links */
-	.igem_2017_content_wrapper a {
-		font-weight: bold;
-		text-decoration: underline;
-		text-decoration-color: #3399ff;
-		color:  #3399ff;
-		-webkit-transition: all 0.4s ease;
-		 -moz-transition: all 0.4s ease;
-		-ms-transition: all 0.4s ease;
-		-o-transition: all 0.4s ease;
-		transition: all 0.4s ease;
-	}
-	/* hover for the links */
-	.igem_2017_content_wrapper a:hover {
-		text-decoration:none;
-		color:#000000;
-	}
-	/* non numbered lists */
-	.igem_2017_content_wrapper ul {
-		padding:0px 20px;
-		font-size: 13px;
-		font-family:Tahoma, Geneva, sans-serif;
-	}
-	/* numbered lists */
-	.igem_2017_content_wrapper ol {
-		padding:0px;
-		font-size: 13px;
-		font-family:Tahoma, Geneva, sans-serif;
-	}
-	/* Table */
-	.igem_2017_content_wrapper table {
-		width: 97%;
-		margin:15px 10px;
-		border: 1px solid #383838;
-		border-collapse: collapse;
-	}
-	/* table cells */
-	.igem_2017_content_wrapper  td {
-		padding: 10px;
-		vertical-align: text-top;
-		border: 1px solid #383838;
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-	/* table headers */
-	.igem_2017_content_wrapper th {
-		padding: 10px;
-		vertical-align: text-top;
-		border: 1px solid #383838;
-		border-collapse: collapse;
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-/**********************************LAYOUT CLASSES **********************************/
-	/* general class for column divs */
-	.igem_2017_content_wrapper .column  {
-		padding: 10px 0px;
-		float:left;
-	}
-	/* class for a full width column */
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-		width:100%;
-	}
-	/* styling for images in a full width column*/
-	.column.full_size img {
-		width:97%;
-		padding: 10px 15px;
-	}
-	/* class for a half width column */
-	.column.half_size {
-		width: 50%;
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-	/* styling for images in a half width column*/
-	.column.half_size img {
-		width: 94.5%;
-		padding: 10px 15px;
-	}
-/********************************* SUPPORT CLASSES ********************************/
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-	.igem_2017_content_wrapper .clear {
-		clear:both;
-	}
-	/* adds extra spacing when clearing content */
-	.igem_2017_content_wrapper  .clear.extra_space {
-		height: 30px;
-	}
-	/* highlight class, makes content slightly smaller */
-	.igem_2017_content_wrapper .highlight {
-		margin: 0px 15px;
-		padding: 15px 0px;
-	}
-	/* highlight class, adds a gray background */
-	.igem_2017_content_wrapper .highlight.gray {
-		background-color: #7c6429;
-	}
-	/* highlight with decoration blue line on top */
-	.igem_2017_content_wrapper .highlight.blue_top {
-    	border-top: 4px solid #3399ff;
-	}
-	/* highlight with a full blue border decoration */
-	.igem_2017_content_wrapper .highlight.blue_border {
-    	border: 4px solid  #3399ff;
-	}
-	/* button class */
-	.igem_2017_content_wrapper .button{
-		max-width: 35%;
- 		margin: 30px auto;
-		padding: 12px 10px;
-     	background-color: #3399ff;
-    	text-align: center;
-   		color: #ffffff;
-		-webkit-transition: all 0.4s ease;
-		-moz-transition: all 0.4s ease;
-		-ms-transition: all 0.4s ease;
-		-o-transition: all 0.4s ease; transition: all 0.4s ease;
-	}
-	/* styling for button on hover */
-	.igem_2017_content_wrapper .button:hover{
-		background-color: #3399ff;
-    	color: #000000;
-	}
-/***************************************************** RESPONSIVE STYLING ****************************************************/
-	/* IF THE SCREEN IS LESS THAN 1200PX */
-	@media only screen and (max-width: 1200px) {
-		#content {width:100%; }
-		.igem_2017_menu_wrapper {width:15%; right:0;}
-		.highlight {padding:10px 0px;}
-		.igem_2017_menu_wrapper #display_menu_control { display:none; }
-		#menu_content { display:block;}
-		.menu_button.direct_to_page {padding-left: 17px;}
-	}
-	/* IF THE SCREEN IS LESS THAN 800PX */
-	@media only screen and (max-width: 800px) {
- 		.igem_2017_menu_wrapper { width:100%; height: 15%; position:relative; left:0%;}
-		.igem_2017_content_wrapper {width:100%; margin-left:0px;}
-		.column.half_size  {width:100%; }
-		.column.full_size img, .column.half_size img {  width: 100%; padding: 10px 0px;}
-		.highlight {padding:15px 5px;}
-		.igem_2017_menu_wrapper #display_menu_control { display:block; }
-		#menu_content { display:none;}
-		.igem_2017_menu_wrapper .menu_button .expand_collapse_icon { width: 5%; }
-		.menu_bottom_padding {display:none;}
-		.menu_button.direct_to_page { padding-left: 36px; }
-	}
-	/* special class that the system uses to make sure the team wants a page to be evaluated */
-	.judges-will-not-evaluate {
-    	width: 96.6%;
-  		margin: 5px 15px;
-  		display: block;
-		border: 4px solid #3399ff;
-    	font-weight: bold;
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-</style>
-<div class="igem_2017_menu_wrapper" >
-	<!-- this div is hidden by default and will only be displayed if the screen size is too small -->
-	<div class="menu_button" id="display_menu_control">
-		MENU
-	</div>
-	<div id="menu_content">
-			<a href="https://2017.igem.org/Team:Cadets2Vets">
-			<div class="menu_button direct_to_page">
-				HOME
-			</div>
-		</a>
-		<div class="menu_button">
-			<div class="expand_collapse_icon">  </div> TEAM
-		</div>
-		<div class="submenu_wrapper" id="team_submenu">
-					<a href="https://2017.igem.org/Team:Cadets2Vets/Team">
-				<div class="submenu_button" id="Team_page">
-					Team
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Collaborations">
-				<div class="submenu_button"  id="Collaborations_page">
-					Collaborations
-				</div>
-			</a>
-		</div>
-		<div class="menu_button">
-			<div class="expand_collapse_icon">  </div> PROJECT
-		</div>
-		<!-- project submenu -->
-		<div class="submenu_wrapper">
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Description">
-				<div class="submenu_button"  id="Description_page">
-					Description
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Design">
-				<div class="submenu_button"  id="Design_page">
-					Design
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Experiments">
-				<div class="submenu_button"  id="Experiments_page">
-					Experiments
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Notebook">
-				<div class="submenu_button"  id="Notebook_page">
-					Notebook
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/InterLab">
-				<div class="submenu_button"  id="InterLab_page">
-					InterLab
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Contribution">
-				<div class="submenu_button"  id="Contribution_page">
-					Contribution
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Model">
-				<div class="submenu_button"  id="Model_page">
-					Model
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Results">
-				<div class="submenu_button"  id="Results_page">
-					Results
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Demonstrate">
-				<div class="submenu_button"  id="Demonstrate_page">
-					Demonstrate
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Improve">
-				<div class="submenu_button"  id="Improve_page">
-					Improve
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Attributions">
-				<div class="submenu_button"  id="Attributions_page">
-					Attributions
-				</div>
-			</a>
-		</div>
-		<div class="menu_button">
-			<div class="expand_collapse_icon">  </div> PARTS
-		</div>
-		<!-- parts submenu -->
-		<div class="submenu_wrapper">
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Parts">
-				<div class="submenu_button"  id="Parts_page">
-					Parts
-				</div>
-			</a>
-			<a href="https://2017.igem.org/Team:Cadets2Vets/Basic_Part">
-				<div class="submenu_button"  id="Basic_Part_page">
-					Basic Parts
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