Difference between revisions of "Team:OUC-China/Improve"

Latest revision as of 13:58, 18 November 2017

Improve

This year, our team improved the promoter BBa_J23018 in a novel way. The newly constructed promoter is BBa_K2314212, in which we add a UTRrpsT sequence and enhanced its strength. Considering relative researches and the result of our experiments, we have the reason to believe that UTRrpsT has the potential to enhance the promoter as a universal enhancement module.

Inspiration

As known to all, 5'UTR will be transcribed and can be used as a regulatory element to adjust the translation process. Therefore, it is believed that a delicate structure of the 5'UTR has the potential as a universal enhancement module. We see that Zhou et al. screened some 5'UTR sequences from E. coli and helped improve the expression of target protein. Thinking about our need to express target gene in our project, we decided to edit an existing promoter in Part in this way. Hopefully, the strength will be enhanced. Since the promoter we picked was not operated this way before, we were not sure about its result at first, so we consulted the auther of our basic paper. Luckily,the idea also received the author's support.[1]

Design

We selected BBa_J23108 from constitutive promoter family and added a 5'UTR sequence that we choose from the paper mentioned above thereafter. Fluorescent protein is used to characterize whether or not the 5'UTR has an enhanced effect on the promoter.

Proof of concept

We constructed a circuit devices with a commonly used red fluorescent protein and randomly selected the promoter and the terminator. The difference is that UTRrpsT is added to a loop and the fluorescence intensity is measured using a microplate reader and plotted. We found that the fluorescence intensity at 6h increased to 1.5 fold. This indicates that UTRrpsT has the capability to function as a generic enhancement module.（Error bars represent standard deviation of three biological replicates.）

Future work

According to the analysis of data, we may draw a conclusion that the part can impove the expression of our target protein (mRFP). In the future, we can change different reporters to detect whether different reporters may have the same result---that their expression will all be improve. If so, we can make sure that the part is universal for improving all proteins and we can utilize this provement to increase the expression of mSA, which improve the probability of binding to yeast successfully and strengthen the binding. Further, we might offer help to other teams.

Reference

[1]Zhou, Shenghu, et al. "Obtaining a Panel of Cascade Promoter-5′-UTR Complexesin Escherichia coli." Acs Synthetic Biology 6.6(2017)

@@ Line 6: / Line 6: @@
 <title>Improve</title>
 <style type="text/css">
-.ouc-right{position: relative;}
+	.ouc-right{position: relative;}
-	.ouc-rightnav{position:absolute;right: -200%;top: 0; display: none;background-color: white;width: 200%;}
+	.ouc-rightnav{position:absolute;right: -200%;top: 0; display: none;background-color: white;width: 200%; z-index: 2001}
 	.ouc-right:visited{background-color:#D2D2D2; color: white;text-decoration: none;}
 	.ouc-right:hover .ouc-rightnav{display: inherit;}
@@ Line 13: / Line 13: @@
 	.ouc-rightnav a:visited {color: #008EA1;text-decoration: none; }
 	.ouc-rightnav a:hover {background-color:#D2D2D2; color: white;text-decoration: none; }
 .ouc-vicedown{position: absolute; top:50px; }
 	.ouc-vicedown a{display: block;height: 40px; width: 100%;text-decoration: none;color: #008EA1; line-height: 40px;text-align: center;}
 	.ouc-vicedown a:visited{color:#008EA1;text-decoration: none }
-	.ouc-vicedown a:hover{color:  #EDEC8C}
+	.ouc-vicedown a:hover{color:  #EDEC8Cl}
-	.ouc-nav{color:white; background-color: #008EA1;display:block; width: 100%; color: white;}
+	.ouc-nav{color:white; background-color: #008EA1;display:block; width: 100%; color: white;padding: 0}
 	.ouc-nav:visited{text-decoration: none; color: white}
 	.ouc-nav:hover{text-decoration: none; background-color: white; color:#008EA1; }
 	.ouc-navbar{position: relative;padding: 0;margin: 0;}
 	.ouc-navbar:hover .ouc-down{display: inherit;}
-	.ouc-down{position: absolute; top:50px;display: none;background-color: white;}
+	.ouc-down{position: absolute; top:50px;display: none;background-color: white; z-index: 1001}
 	.ouc-navdown{color: #008EA1;display: block; width: 100%;height: 40px;}
 	.ouc-navdown:visited{text-decoration: none;color:#008EA1; }
-	.ouc-navdown:hover{background-color:#D2D2D2;color: white;text-decoration: none}
+	.ouc-navdown:hover{background-color:#D2D2D2;color: white;text-decoration: none; z-index: 1501}
-	.ouc-guide{position: fixed; width: 100%;top: 15px;z-index: 1000}
+	.ouc-guide{position: fixed; width: 100%;top: 15px;z-index:1000}
 	.ouc-page-header{border-bottom-color: #EDEC8C;border-bottom-width: thick;color: #008F75;}
 	.top{position: fixed;bottom:0;right: 0;}
 	.top:hover{animation: upp 0.6s ;}
 	@keyframes upp {
 % {margin-bottom: 0;}
@@ Line 37: / Line 39: @@
 	}
 	.ouc-reserve{background-color: #008EA1;color: white;}
-	.ouc-heading {color: #008EA1;}
+    .ouc-heading {margin: 0;color: #008EA1;}
-	.ouc-a{color:#E3D434; }
-	.ouc-a:hover{color: #008F75;}
-	.ouc-ul{border-left:thin dashed #008F75}
-	.ouc-panel-heading{text-align: center; color:#008EA1; }
 </style>
 </head>
 <body>
@@ Line 50: / Line 47: @@
 <div class="ouc-guide" style="height: 50px;background-color:#008EA1 ;">
 	<div class="row">
-	    <div class="col-md-1"></div>
-		<div class="col-md-1" style="padding-top: 5px;"><a href="https://2017.igem.org/Team:OUC-China"><img src="https://static.igem.org/mediawiki/2017/8/89/T--OUC-China--logo.jpg" height="40px"/></a></div>
+		<div class="col-md-2 navbar-header" style="text-align: center; padding: 0">
-		<div class="col-md-1"></div>
-		<div class="col-md-1 ouc-navbar">
+		<button class="navbar-toggle" type="button" data-toggle="collapse" data-target=".navbar-responsive-collapse">
+         <span class="sr-only">Toggle Navigation</span>
+         <span class="caret" style="color: white"></span>
+       </button>
+		<a href="https://2017.igem.org/Team:OUC-China"><img src="https://static.igem.org/mediawiki/2017/8/89/T--OUC-China--logo.jpg" height="50px"/></a></div>
+		<div class="collapse navbar-collapse navbar-responsive-collapse" style="padding: 0">
+		<div class="col-md-1" style="padding: 0"></div>
+		<div class="col-md-1 ouc-navbar" style="padding: 0">
 		    <a href="##" style="line-height: 50px;text-align: center" class="ouc-nav">Team<span class="caret"></span></a>
 		    <div class="ouc-down" style=" width: 100%; text-align: center">
@@ Line 61: / Line 71: @@
 		    </div>
 		</div>
-		<div class="col-md-1 ouc-navbar">
+		<div class="col-md-1 ouc-navbar" style="padding: 0">
 		    <a href="##" style="line-height: 50px;text-align: center" class="ouc-nav">Project<span class="caret"></span></a>
 		    <div class="ouc-down" style=" width: 100%; text-align: center">
 		    	<div><a href="https://2017.igem.org/Team:OUC-China/Description" style="line-height: 40px; " class="ouc-navdown">Description</a></div>
 		    	<div><a href="https://2017.igem.org/Team:OUC-China/Design" style="line-height: 40px; " class="ouc-navdown">Design</a></div>
-<div class="ouc-right">
+                <div class="ouc-right">
-		    	    <a style="line-height: 40px; font-size: 15px;" class="ouc-navdown">Proof of concept</a>
+		    	    <a style="line-height: 40px; font-size: 15px;" class="ouc-navdown" href="https://2017.igem.org/Team:OUC-China/proof1">Proof of concept</a>
 		    	    <div class="ouc-rightnav">
-		    	    	<a href="" style="display: block">Basic fermentation</a>
+		    	    	<a href="https://2017.igem.org/Team:OUC-China/proof1" style="display: block">Basic fermentation</a>
-		    	    	<a href="" style="display: block">Co-express platform</a>
+		    	    	<a href="https://2017.igem.org/Team:OUC-China/proof2" style="display: block">Adhesion platform</a>
-		    	    	<a href="" style="display: block">Mini combination</a>
+		    	    	<a href="https://2017.igem.org/Team:OUC-China/proof3" style="display: block">MINI-GRE</a>
 		    	    </div>
 		    	</div>
-		    	<div><a href="https://2017.igem.org/Team:OUC-China/Demonstrate" style="line-height: 40px; " class="ouc-navdown">Demonstrate</a></div>
+		    	<div><a href="https://2017.igem.org/Team:OUC-China/Demonstrate" style="line-height: 40px; " class="ouc-navdown">Demonstration</a></div>
 		    	<div><a href="https://2017.igem.org/Team:OUC-China/InterLab" style="line-height: 40px; " class="ouc-navdown">InterLab</a></div>
-		    	<div><a href="https://2017.igem.org/Team:OUC-China/Improve" style="line-height: 40px; " class="ouc-navdown">Improve</a></div>
+		    	<div><a href="https://2017.igem.org/Team:OUC-China/Improve" style="line-height: 40px; " class="ouc-navdown">Improvement</a></div>
 		    	<div><a href="https://2017.igem.org/Team:OUC-China/Notebook" style="line-height: 40px; " class="ouc-navdown">Notebook</a></div>
 		    </div>
 		</div>
-		<div class="col-md-1"><a href="https://2017.igem.org/Team:OUC-China/Model" style="line-height: 50px;text-align: center" class="ouc-nav">Model</a></div>
+		<div class="col-md-1" style="padding: 0"><a href="https://2017.igem.org/Team:OUC-China/Model" style="line-height: 50px;text-align: center" class="ouc-nav">Model</a></div>
-		<div class="col-md-1"><a href="https://2017.igem.org/Team:OUC-China/Parts" style="line-height: 50px;text-align: center" class="ouc-nav">Parts</a></div>
+		<div class="col-md-1" style="padding: 0"><a href="https://2017.igem.org/Team:OUC-China/Parts" style="line-height: 50px;text-align: center" class="ouc-nav">Parts</a></div>
-		<div class="col-md-1"><a href="https://2017.igem.org/Team:OUC-China/Safety" style="line-height: 50px;text-align: center" class="ouc-nav">Safety</a></div>
+		<div class="col-md-1" style="padding: 0"><a href="https://2017.igem.org/Team:OUC-China/Safety" style="line-height: 50px;text-align: center" class="ouc-nav">Safety</a></div>
-		<div class="col-md-2 ouc-navbar">
+		<div class="col-md-2 ouc-navbar" style="padding: 0">
-		    <a href="##" style="line-height: 50px;text-align: center" class="ouc-nav">Human Practice<span class="caret"></span></a>
+		    <a href="https://2017.igem.org/Team:OUC-China/Engagement" style="line-height: 50px;text-align: center" class="ouc-nav">Human Practice<span class="caret"></span></a>
 		    <div class="ouc-down" style=" width: 100%; text-align: center">
 		    	<div><a href="https://2017.igem.org/Team:OUC-China/HumanPractice" style="line-height: 40px; " class="ouc-navdown">Overview</a></div>
@@ Line 92: / Line 102: @@
 		    </div>
 		</div>
-		<div class="col-md-2"></div>
+		<div class="col-md-2" style="padding: 0"></div>
+		</div>
 	</div>
 </div>
 <!--此处是导航-->
@@ Line 100: / Line 117: @@
 <div class="container">
      <div class="page-header ouc-page-header">
-         <h1><strong>Improve</strong></h1>
+         <h1><strong>Improvement</strong></h1>
      </div>
-     <h3 class="ouc-heading"><strong>Inspiration</strong></h3>
+<p style="font-size: 20px">
+      This year, our team improved the promoter <a href = "http://parts.igem.org/Part:BBa_J23108" style = “color:blue;">BBa_J23018</a> in a novel way. The newly constructed promoter is <a href = "http://parts.igem.org/Part:BBa_K2314212" style = “color:blue;">BBa_K2314212</a>, in which we add a UTRrpsT sequence and enhanced its strength. Considering relative researches and the result of our experiments, we have the reason to believe that UTRrpsT has the potential to enhance the promoter as a universal enhancement module.
+     <h2 class="ouc-heading"><strong>Inspiration</strong></h2>
      <div class="row">
      	<div class="col-md-4" style="text-align: center;">
@@ Line 111: / Line 132: @@
      		<p style="font-size: 20px">
      		<br/><br/><br/><br/>
-'UTR will be transcribed and can be used as a regulatory element to adjust
+     			As known to all, 5'UTR will be transcribed and can be used as a regulatory element to adjust
-the translation process. Thus, there is reason to believe that a delicate
+the translation process. Therefore, it is believed that a delicate
 structure of the 5'UTR has the potential as a universal enhancement
-module. We see that Zhou et al. Screened some 5'UTR sequences from <i>E.
+module. We see that Zhou et al. screened some 5'UTR sequences from <i>E.
-coli</i>, which helped to improve protein content. Thus, we are prepared to
+coli</i> and helped improve the expression of target protein. Thinking about our need to express target gene in our project, we decided to edit an existing promoter in Part in this way. Hopefully, the strength will be enhanced. Since the promoter we picked was not operated this way before, we were not sure about its result at first, so we consulted the auther of our basic paper. Luckily,the idea also received the
-consider this part as an enhancement module, the idea also received the
 author's support.[1]
      		</p>
@@ Line 122: / Line 142: @@
      </div>
-     <h3 class="ouc-heading"><strong>Design</strong></h3>
+     <h2 class="ouc-heading"><strong>Design</strong></h2>
      <p style="text-align: center">
      	<img src="https://static.igem.org/mediawiki/2017/thumb/a/aa/T--OUC-China--improve.illustration.png/800px-T--OUC-China--improve.illustration.png" width="700px"/>
      </p>
      <p style="font-size: 20px">
-     	We selected J23108 from constitutive
+     	We selected BBa_J23108 from constitutive
-promoter family members and added the
+promoter family  and added a
-'UTR sequence thereafter. Fluorescent
+'UTR sequence that we choose from the paper mentioned above thereafter. Fluorescent
-protein is used to characterize whether the
+protein is used to characterize whether or not the
 'UTR has an enhanced effect on the
 promoter.
      </p>
-     <h3 class="ouc-heading"><strong>Proof of concept</strong></h3>
+     <h2 class="ouc-heading"><strong>Proof of concept</strong></h2>
      <p style="text-align: center">
      	<img src="https://static.igem.org/mediawiki/2017/thumb/7/77/T--OUC-China--improve.FI_Abs600.png/800px-T--OUC-China--improve.FI_Abs600.png" width="700px"/>
      </p>
      <p style="font-size: 20px">
-     	We constructed two red fluorescent protein devices and constructed the loop by randomly
+     	We constructed a circuit devices with a commonly used red fluorescent protein and  randomly
-selecting the promoter and the terminator. The difference is that UTRrpsT is added to a loop and
+selected the promoter and the terminator. The difference is that UTRrpsT is added to a loop and
 the fluorescence intensity is measured using a microplate reader and plotted. We found that the
-fluorescence intensity of 6h increased by 1.5 fold. This indicates that UTRrpsT has the capability
+fluorescence intensity at 6h increased to 1.5 fold. This indicates that UTRrpsT has the capability
-as a generic enhancement module.
+to function as a generic enhancement module.（Error bars represent standard deviation of three biological replicates.）
+<h2 class="ouc-heading"><strong>Future work</strong></h2>
+        <p style="font-size: 20px">
+        According to the analysis of data, we may draw a conclusion that the part can impove the expression of our target protein (mRFP). In the future, we can change different reporters to  detect whether different  reporters may have the same result---that their expression will all be improve. If so, we can make sure that the part is universal for improving all proteins and we can utilize this provement to increase the expression of mSA, which improve the probability of binding to yeast successfully and strengthen the binding. Further, we might offer help to other teams.
      </p>
-     <h3 class="ouc-heading"><strong>Reference</strong></h3>
+     <h2 class="ouc-heading"><strong>Reference</strong></h2>
 [1]Zhou, Shenghu, et al. "Obtaining a Panel of Cascade Promoter-5′-UTR Complexesin Escherichia coli." Acs Synthetic Biology 6.6(2017)<br/>
 </div>