View source for Team:Tokyo Tech/AHL Assay/AHL Reporter Assay

{{Tokyo_Tech}}
<html>
<head>
	<style type="text/css">
<!--
/********************** All style of this page ************************/
body{
    background-image: url("https://static.igem.org/mediawiki/2016/8/83/Tokyo_Tech_Background.png");
    background-repeat: no-repeat;
    background-position: center center;
    background-size: cover;
    background-attachment: fixed;
}
#globalWrapper, #content{
    background: transparent;
}
#content{
//    background-image: url("https://static.igem.org/mediawiki/2016/8/83/Tokyo_Tech_Background.png");
    margin: 0 auto;
}
/**********************************************************************/
#main_contents{
    margin: 0 auto;
    width: 90%;
    min-width: 880px;
    max-width: 1100px;
}
.container{
    margin: 5px auto;
    background: #ffffff;
    border: 1px solid #583612;
}
.container_top h1{
	margin-top: 1px;
	margin-bottom: 1px;
	height: 38px;
	font-size: 36px;
}
.container_header{
    background: #ffffff;
    text-align: center;
}
.container_header span{
    border-bottom: 2px solid #583612;
}
.container_contents{
	line-height: 1.5em;
	width: 800px;
	margin: 0 auto;
	padding-bottom: 10px;
}
#main_contents p.normal_text{
	font-size: 18px;
	text-indent: 18px;
	text-align: justify;
//	padding: 18px;
    font-family: 'Century', serif;
}

#
/************************* Show/Hide setting ***************************/
.on{
	display: block;
	-webkit-transiton: all 0.6s ease;
	transition: all 0.6s ease;
}
.off{
	display: none;
	-webkit-transiton: all 0.6s ease;
	transition: all 0.6s ease;
}

/************************* This page setting ******************************/
#contents_menu{
	display: inline-block;
	border: 3px double #ffffff;
}
--></style>
<script src="https://2015.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML"></script>
	<script type="text/x-mathjax-config">
		MathJax.Hub.Config({
			TeX: { equationNumbers: {autoNumber: "AMS"} },
			displayAlign: "left",
			displayIndent: "2em"
		});
	</script>
</head>
<body>
	<div id="main_contents">
		<div id="page_header" class="container container_top">
			<h1 align="center">3-2-1 AHL reporter assay</h1>
		</div><!-- /page_header -->
		<div id="contents" class="container">
			<div id="contents_header" class="container_header">
				<h2><span>Contents</span></h2>
			</div><!-- /contents_header -->
			<div id="contents_contents" class="container_contents">
				<div id="contents_menu">
				<h3 class="link"><a href="#introduction">1. Introduction</a></h3>
				<h3 class="link"><a href="#summary">2. Summary of the experiment</a></h3>
				<h3 class="link"><a href="#results">3. Results</a></h3>
				<h3 class="link"><a href="#discussion">4. Discussion</a></h3>
				<h3 class="link"><a href="#methods">5. Materials and methods</a></h3>
				<h3 class="link"><a href="#construction"><font size="2.7">&nbsp;&nbsp;&nbsp;5-1. Construction</font></a></h3>
				<h3 class="link"><a href="#protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;5-2. Assay protocol</font></a></h3>
				<h3 class="link"><a href="#reference">6. Reference</a></h3>
				</div><!-- /contents_menu -->
			</div><!-- /contents_contents -->
		</div><!-- /contents -->

		<div id="introduction" class="container">
			<div id="introduction_header" class="container_header">
				<h2><span>1. Introduction</span></h2>
			</div><!-- /_header -->
			<div id="introduction_contents" class="container_contents">
				<p class="normal_text"> This section describes the evaluation of the module of Quorum Sensing. Snow White seems to die after eating the Poisoned Apple that was given by Queen. In order to trace this scene with <i>E. coli</i>, establishing cell-cell communication is necessary. <br><br>AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing” system. In our project, we decided to use three AHLs studied well by several preceding iGEM Teams; the AHL molecules used here were C4HSL (hereafter referred to as C4), 3OC6HSL (C6), and 3OC12HSL (C12). Using these molecules, we enable the <i>E. coli</i> cells in a test tube to communicate and interact. By the experiments in this section, the module of Quorum Sensing was evaluated.
				</p>
				
			</div><!-- /introduction_contents -->
		</div><!-- /introdution -->

		<div id="summary" class="container">
			<div id="id_header" class="container_header">
				<h2><span>2. Summary of the experiment</span></h2>
			</div><!-- /_header -->
			<div id="summary_contents" class="container_contents">
				<p class="normal_text"> The purpose of the experiments in this section is to evaluate the activities of Prhl (<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017</a>), Plux (<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062</a>), and Plas (<a href="http://parts.igem.org/Part:BBa_R0079">BBa_R0079</a>) promoters upon addition of different AHLs. When C4, C6, and C12 internalize into <i>E. coli</i> cells, these molecules are accepted by the corresponding receptor proteins, RhlR, LuxR, and LasR, respectively. Then, the RhlR-C4, LuxR-C6, and LasR-C12 complexes activate/repress transcription from the Prhl, Plux, and Plas, respectively. However, it should be considered that illegitimate reactions of an AHL to non-native receptor proteins also occur to some extent. This phenomenon is called 'crosstalk.' We wanted to seek the best combination of AHL systems for a co-culture of Snow White <span style ="font-style : italic"> coli</span> and Queen <span style="font-style : italic">coli</span>. To this end, four plasmids shown below were constructed and the Relative Fluorescent Unit (RFU) of GFP/ Turbidity after adding each AHL to the culture medium was measured.  
				</p>
<br>
<br>
<br>

<p class="normal_text"> -Plasmids<br>

					<p class="normal_text"> A. Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">rhlR</span>(<a href="http://parts.igem.org/Part:BBa_C0071">BBa_C0071)</a>-<span style ="font-style : italic">tt</span>-Prhl(<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017)</a>-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) (Fig. 3-2-1-2-1)

					</p><br>
					<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/72/FIg.3-2-1-5-1.png" height ="150"><br></div>
					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-1-2-1 </span> Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">rhlR</span>-<span style ="font-style : italic">tt</span>-Prhl-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1)
					</p></div><br>

					<p class="normal_text">B. Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">luxR</span>(<a href="http://parts.igem.org/Part:BBa_C0062">BBa_C0062)</a>-<span style ="font-style : italic">tt</span>-Plux(<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062)</a>-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) (Fig. 3-2-1-2-2)</p><br><br>
					<div align="center"><img src="https://static.igem.org/mediawiki/2016/e/e5/Fig._3-2-1-5-2.png" height ="150"><br></div>
					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-1-2-2 </span> Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">rhlR</span>-<span style ="font-style : italic">tt</span>-Prhl-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1)
					</p></div><br>
					<p class="normal_text"> C. Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">lasR</span>(<a href="http://parts.igem.org/Part:BBa_C0179">BBa_C0179)</a>-<span style ="font-style : italic">tt</span>-Plas(<a href="http://parts.igem.org/Part:BBa_R0079">BBa_R0079)</a>-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pBS6A1) (Fig. 3-2-1-2-3)</p><br>
					<div align="center"><img src="https://static.igem.org/mediawiki/2016/archive/c/c8/20161018135157%21Fig._3-2-1-5-3.png" height ="150"><br></div>
					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-1-2-3 </span> Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">lasR</span>-<span style ="font-style : italic">tt</span>-Plas-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pBS6A1)</p></div><br>

					<p class="normal_text"> D. pSB6A1 …Nagative control (Fig. 3-2-1-2-4)</p><br>
					<div align="center"><img src="https://static.igem.org/mediawiki/2016/5/5c/Fig._3-2-1-5-4.png" height ="150"><br></div>
					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-1-2-4 </span> pSB6A1</p></div>



			</div><!-- /summary_contents -->
		</div><!-- /summary -->
		
		<div id="results" class="container">
			<div id="results_header" class="container_header">
				<h2><span>3. Results</span></h2>
			</div><!-- /_header -->
			<div id="results_contents" class="container_contents">
				<p class="normal_text"> The experiments proved that our parts worked as expected (Fig.3-2-1-3).<br>
				 The ratio of promoter strength is shown below.
<br> Prhl(<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017)</a> : Plux(<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062)</a> : Plas(<a href="http://parts.igem.org/Part:BBa_R0079">BBa_R0079)</a>= 2 : 40 :1<br>
				The Lux system, which is usually activated by C6, reacted not only to C6 but also to C12 (crosstalk). The reaction rate to C12 was half as that to C6. The Rhl system, which is usually activated by C4, reacted not only to C4 but also to C6 (crosstalk). The reaction rate to C6 was 0.78-fold relative to that to C4.
				</p>
				<div align="center"><img src="https://static.igem.org/mediawiki/2016/c/cd/Fig.3-2-1-3.png" height =""><br></div>
					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-1-3 </span> RFU of GFP/ Turbidity of AHL Reporter Assay
					</p></div>
			</div><!-- /results_contents -->
		</div><!-- /results -->
		
		<div id="discussion" class="container">
			<div id="discussion_header" class="container_header">
				<h2><span>4. Discussion</span></h2>
			</div><!-- /_header -->
			<div id="discussion_contents" class="container_contents">
				<p class="normal_text"> Considering different expression level from the AHL-inducible promoters shown above, we should find and choose the appropriate combination of the AHL molecules, the receptor proteins, and the AHL-inducible promoters for our final genetic circuits. The above results implied that, for expression induction from Plux(<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062)</a>, using not C6 but C12 was better to orchestrate the promoter activities. However, the promoter activity of Prhl(<a href"=http://parts.igem.org/Part:BBa_I14017">BBa_I14017)</a> was still estimated to be incomparable to that of Plux(<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062)</a>. In fact, when our final circuits was simulated, it was found that the Prhl(<a href="http://parts.igem.org/Parts:BBa_I14017">BBa_I14017)</a> activity was too weak compared to Plux(<a href="http://parts.igem.org/Part:BBa_R0062">BBa_R0062)</a> to operate our final circuits successfully (see the Model page). Taken together, we concluded that improvement of Prhl(<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071)</a> was necessary to increase its promoter activity and orchestrate the promoter activities (see the Rhl system assay page).
				</p>
			</div><!-- /discussion_contents -->
		</div><!-- /discussion -->
		
		<div id="methods" class="container">
			<div id="methods_header" class="container_header">
				<h2><span>5. Materials and methods</span></h2>
			</div><!-- /_header -->
			<div id="methods_contents" class="container_contents">
			<div id="construction">			
				<div id="construction_header">
					<h3><span>5-1. Construction</span></h3>
				</div><!-- /_header -->
				<div id="construction_contents">
					<p class="normal_text"> -Strain<br>
 					  All the plasmids were prepared in XL1-Blue strain.<br><br>

					
					<p class="normal_text">-Medium<br>
 					LB medium A:<br>
 					   LB medium containing ampicillin (50 microg/ mL) 
					</p>
				</div><!-- /constrution_contents -->			
			</div><!-- /construction -->	
<br><br><br><br>
			<div id="protocol">	
				<div id="protocol_header">
					<h3><span>5-2. Assay protocol</span></h3>
				</div><!-- /_header -->			
				<div id="methods_contents">
					<p class="normal_text">The following experiments are performed at 37℃ unless otherwise stated.<br><br>

 					1) Prepare overnight cultures for each sample in 3 mL LB medium A with vigorous shaking.<br><br>
 
 					2) Dilute the overnight cultures to 1 / 60 in fresh LB medium A (1.2 mL).<br><br>

 					3) Incubate the fresh cultures for 1 h with vigorous shaking.<br><br>
 
 					4) Add 500 microM C4, 500 microM C6, 500 microM C12 or DMSO to each 500 microL sample at the final concentration 10 microM.<br><br>
 
 					5) Incubate the samples for 4 h with vigorous shaking.<br><br>
 
 					6) Add 100 microL of the samples to each well of a plate reader.<br><br>
 
 					7) Measure RFU of GFP  at 490 nm as an exciting wavelength, 525 nm as an emission wavelength.    <br><br>
 
 					8) Measure the turbidity at 600 nm.

					</p>
				</div><!-- /protocol_contents -->				
			</div><!-- /protocol -->
			</div><!-- /methods_contents -->
		</div><!-- /methods -->
		
		<div id="reference" class="container">
			<div id="reference_header" class="container_header">
				<h2><span>6. Reference</span></h2>
			</div><!-- /_header -->
			<div id="reference_contents" class="container_contents">
				<p class="normal_text"><br>(1) Kendall M. Gray et al. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of <span style ="font-style : italic">Vibrio fischeri</span> and <span style ="font-style : italic">Pseudomonas aeruginosa</span>. Journal of Bacteriology 176(10): 3076–3080
 

				</p>
			</div><!-- /reference_contents -->
		</div><!-- /reference -->


</body>
</html>