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			<h1 align="center">3-2-3 Rhl system Assay</h1>
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				<h2><span>Contents</span></h2>
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				<h3 class="link"><a href="#introduction">1. Introduction</a></h3>
				<h3 class="link"><a href="#summary">2. Summary of the experiments</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>
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				<h2><span>1. Introduction</span></h2>
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				<p class="normal_text">
                                 As shown in the previous sections, Prhl(<a href="http://parts.igem.org/Part:BBa_I14017">BBa_I14017</a>) activity was too weak compared to Plux to operate the final genetic circuits. Similarly, simulation results suggested that the circuits did not work as we desired due to the same reason (see the Model page). We expected that this situation can be circumvented by using the previously improved Prhl (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>, <a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) that was established by iGEM 2014 Tokyo_Tech Team. However, these Prhl promoters were also found to be inappropriate for two reasons (see the Discussion). Therefore, we improved the Prhl Promoter by our own team and succeeded to obtain novel and appropriate Prhl (Noticeable Mutant) (<a href="http://parts.igem.org/Part:BBa_K1949060">BBa_K1949060</a>), hereafter referred to as Prhl(NM).
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				<h2><span>2. Summary of the experiments</span></h2>
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				<p class="normal_text"> The purpose of the experiment in this section is to create a strong Prhl for our final genetic circuits.<br>
				　This experiment consists of the three parts below. <br>
				&#8544;. Improved Prhl by iGEM 2014 Tokyo_Tech Team and characterization of <span style ="font-style : italic">rhlR</span> <br>
				&#8545;. Improvement of the wild type Prhl <br>
				&#8546;. Comparison of the improved Prhl by iGEM 2014 Tokyo_Tech Team to our original improved Prhl <br>

<br>
<br>
<br>

<p class="normal_text">-Plasmids<br>
				<p class="normal_text">&#8544;. Improved Prhl by iGEM 2014 Tokyo_Tech Team and <span style="font-style : italic">rhlR</span> assay <br>
				 <p class="normal_text">A. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span>(<a href="http://parts.igem.org/Part:BBa_C0071">BBa_C0071</a>) (pSB6A1), Prhl(LR)(<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-2-1)<br><br>


					<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/4b/Fig._3-2-3-5-1.png"" height ="150"><br></div>
					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-1 </span>Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1),<br>
 Prhl(LR) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) 
					</p></div><br>
 				
                                <p class="normal_text">B. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span>(<a href="http://parts.igem.org/Part:BBa_C0171">BBa_C0171</a>) (pSB6A1), Prhl(LR) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span>  (pSB3K3) (Fig. 3-2-3-2-2)<br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/8/8a/Fig._3-2-3-5-2.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-2 </span>Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> (pSB6A1), <br>
Prhl(LR) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) 					</p></div><br>

 				<p class="normal_text">C. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(RL)(<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-2-3)<br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/66/Fig._3-2-3-5-3.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-3 </span>Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1),<br>
 Prhl(RL) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3)  
					</p></div><br>

 				<p class="normal_text">D. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> (pSB6A1), Prhl(RL) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-2-4)<br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/7/7a/Fig._3-2-3-5-4.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-4 </span> Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> (pSB6A1), <br>
Prhl(RL) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) 
					</p></div><br>

 				<p class="normal_text">E. pSB6A1, pSB3K3 …Nagative control (Fig. 3-2-3-2-5)<br><br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/3/35/Fig._3-2-3-5-5.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-5 </span>pSB6A1, pSB3K3 
					</p></div><br>

 
				<p class="normal_text">II. Improvment the wild type Prhl <br>
				 <p class="normal_text">A. Prhl(<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) - <span style="font-style : italic">rbs</span> -  <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-2-6)<br><br>

                                        <div align="center"><img src="https://static.igem.org/mediawiki/2016/c/ce/Fig._3-2-3-5-6.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-6 </span>Prhl - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3)					</p></div><br>


 				 <p class="normal_text">B. Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<i>LVA</i> (pSB6A1), Prhl(NM)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span>(<a href="http://parts.igem.org/Part:BBa_K1949060">BBa_ K1949060</a>) (pSB3K3) (Fig. 3-2-3-2-7)<br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/90/Fig._3-2-3-5-7.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-7 </span>Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<i>LVA</i> (pSB6A1), <br>
Prhl(NM)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3)
					</p></div><br>


				 <p class="normal_text">C. Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(M)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-2-8)<br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6d/Fig._3-2-3-5-8.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-8 </span>Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (pSB6A1), <br>
Prhl(M)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3)					</p></div><br>


				 <p class="normal_text">D. Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(WT)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-2-9)<br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/d/d3/Fig._3-2-3-5-9.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-9 </span>Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (pSB6A1), <br>
Prhl(WT)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3)
					</p></div><br>


				 <p class="normal_text">E. pSB6A1, pSB3K3 …Nagative control (Fig. 3-2-3-2-5)<br><br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/3/35/Fig._3-2-3-5-5.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-5 </span>pSB6A1, pSB3K3
					</p></div><br>

 
				<p class="normal_text">III. Comparison the improved Prhl by iGEM 2014 Tokyo_Tech Team to our original improved Prhl<br>
 				 <p class="normal_text">A. Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(LR)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp </span>(pSB3K3) (Fig. 3-2-3-2-1)<br>


					<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/4b/Fig._3-2-3-5-1.png"" height ="150"><br></div>
					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-1 </span>Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (pSB6A1),<br>
 Prhl(LR)-<span style="font-style : italic">rbs</span>- <span style="font-style : italic">gfp</span>(pSB3K3) 
					</p></div><br>
 				

				 <p class="normal_text">B. Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(NM)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-2-7)<br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/90/Fig._3-2-3-5-7.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-7 </span>Pcon-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<i>LVA</i> (pSB6A1), <br>
Prhl(NM)-<span style="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3)
					</p></div><br>


				 <p class="normal_text">C. pSB6A1, pSB3K3…Negative control (Fig. 3-2-3-2-5)<br><br>

                                       <div align="center"><img src="https://static.igem.org/mediawiki/2016/3/35/Fig._3-2-3-5-5.png"" height ="150"><br></div>
                                        <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-2-5 </span>pSB6A1, pSB3K3
					</p></div>

 
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				<h2><span>3. Results</span></h2>
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				<p class="normal_text">&#8544;. Improved Prhl by iGEM 2014 Tokyo_Tech Team and characterization of <span style ="font-style : italic">rhlR</span>  <br>
				   We found that Prhl(RL) (<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) activity was weak and the expression level from this promoter depended on the presence of LVA tag at the C-terminus of RhlR protein (Fig.3-2-3-3-1); LVA-tagged proteins are prone to be degraded by cellular proteases. Note that the <i>LVA</i> tag is one variant of the <i>ssrA</i> tag which is well-known as a protein degradation sequence in <i>E. coli</i>. Prhl(LR) (<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>) activity was strong but unexpectedly reacted with C12 (crosstalk) (Fig.3-2-3-3-4). Since crosstalk with C12 causes a serious problem in the circuits, this promoter could not be used.
				</p><br>
				<div align="center"><img src="URL"><br></div>
				<div align="center"><img src="https://static.igem.org/mediawiki/2016/e/ee/Fig._3-2-3-3-1.png" height ="300"><br></div>
				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-3-1 </span> Effect of RhlR by tagging LVA
				</p></div><br>
				<p class="normal_text"> The colonies of transformants with a <span style ="font-style : italic">rhlR</span> (<a href="http://parts.igem.org/Part:BBa_C0171">BBa_C0171</a>) plasmid looked rough and growth was slow (Fig.3-2-3-3-2-left), while the colonies of transformants with a <span style ="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> (<a href="http://parts.igem.org/Part:BBa_C0071">BBa_C0071</a>) plasmid looked smooth and the growth was normal(Fig.3-2-3-3-2-right). However, the reason for this result is unclear.</p><br>
				<div align="center"><img src="https://static.igem.org/mediawiki/2016/8/88/Fig._3-2-3-3-2.png" height ="300"><br></div>
				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-3-2 </span>The colonies of transformants with a <span style ="font-style : italic">rhlR</span> (left) or a <span style ="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> right)
				</p></div><br>
				<p class="normal_text">	&#8545;. Improvement of the wild type Prhl <br>
 				  By introducing various single point mutations into wild type Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) by PCR, a library of Prhl mutants was generated. The target site for mutation was the -10 region of Prhl, because the sequence, TAAAAA, is far from the consensus sequence of <i>E. coli</i> -10 region, TATAAT. Then, stronger Prhl was screened from the library, and two Prhl mutants with stronger activity were obtained from 198 mutants tested (Fig.3-2-3-3-3).</p>
				<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/7c/Fig._3-2-3-3-3.png" height ="300"><br></div>
				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-3-3 </span>RFU of GFP / Turbidity of imoroved Prhl mutants
				</p></div>	 				
				<p class="normal_text">The sequences of these two mutants are shown below. The small characters indicate the scar sequence and a single point mutation is colored with red.<br><br>

				Wild type Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) <br>
				TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTAAAAAGTGTTC tactagagAAAGAGGAGAAA <br><br> 

				Prhl(NM) (<a href="http://parts.igem.org/Part:BBa_K1949060">BBa_K1949060</a>)<br>
				TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTA<font color="red">T</font>AAAGTGTTC tactagagAAAGAGGAGAAA <br><br> 
	
				Prhl(M)   <br>                  
				TCCTGTGAAATCTGGCAGTTACCGTTAGCTTTCGAATTGGCTA<font color="red">T</font>AAAGTGTTC tactag<font color="red">ta</font>AAAGAGGAGAAA  <br><br>                  

				&#8546;. Comparison of the improved Prhl by iGEM 2014 Tokyo_Tech Team to our original improved Prhl<br>
				   The activity of Prhl(NM) was compared to Prhl(LR) by observing expression of the reporter gene, (<i>gfp</i>). (Fig.3-2-3-3-4). The reaction activity of Prhl(NM) to C4 was stronger than that of Prhl(LR), and Prhl(NM) did not react with C12 at all.</p>
				<div align="center"><img src="https://static.igem.org/mediawiki/2016/3/33/Fig._3-2-3-3-4.png" height ="300"><br></div>
				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-3-3-4</span> Comparison of Prhl(NM) by Prhl(LR)
				</p></div>				 
		
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				<h2><span>4. Discussion</span></h2>
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			<div id="discussion_contents" class="container_contents">
				<p class="normal_text"> Judging from the result of the experiment I, Prhl(RL) activity was unexpectedly weak and significant expression was obtained only when extremely high C4 concentration, which is employed by iGEM 2014 Tokyo_Tech Team. Such feature of Prhl(RL) is inappropriate for our experiments, because such high level secretion of C4 is not desired from the Snow White-<i>coli</i>.<br>
				In the absence of C4, RhlR is known to repress Prhl and the repression is released when RhlR with bind C4(1). In our experiments, RhlR without LVA tag seemed to repress Prhl very tightly, and as a consequent, GFP could not be expressed very much under low C4 concentrations (Fig.3-2-3-3-1). On the other hand, under high C4 concentrations, it seemed that a lot of RhlR bound with C4, and the repression was successfully released and GFP was expressed greatly(Data not shown). RhlR with LVA tag tends to be degraded easily and its amount in cells should be low at steady-state level. Therefore, in our experiments, the repression by RhlR without LVA tag was not strong and GFP was expressed in high amounts. <br><br>Interestingly, RhlR without the LVA tag repressed growth of <i>E. coli</i>. The reason for this result is unclear, but high RhlR protein level might repress or activate <i>E. coli</i> gene unexpectedly.<br><br>
 				Prhl activity became stronger when the sequence of -10 region was changed from “TAAAAA” to "TATAAA" which is known as that of the strongest promoters in <i>E. coli</i>. Prhl(M) had accidental two mutations in the scar sequence, and it is not suitable for a new part of BioBrick.<br>
 				In our project, the combination of <span style ="font-style : italic">rhlR</span> with <i>LVA</i> tag and Prhl(LR) might work well in our final genetic circuits. However, Prhl(LR) has lux box, and thus, the promoter also reacts with C12 (crosstalk) unintentionally. In our final circuits, we have to use C12 for Plux activation, and thus, usage of Prhl(LR) is inappropriate. Then, we here obtained the Prhl(NM) of which activity was stronger than Prhl(LR). When compared to Prhl(LR), Prhl(NM) showed little crosstalk with C12 and the signal-noise (SN) ratio was higher. The SN ratio of Prhl(LR) to C4 was small, possibly reflecting that leaky expression from Prhl(LR) was high.<br>
 				As a conclusion, the combination of <span style="font-style : italic">rhlR</span> with <i>LVA</i> tag and Prhl(NM) is best for our final genetic circuits.

				</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.</p>

				<p class="normal_text"> -Plasmids<br>
				&#8544;. Improved Prhl by iGEM 2014 Tokyo_Tech Team and <span style="font-style : italic">rhlR</span> assay <br>
				 A. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span>(<a href="http://parts.igem.org/Part:BBa_C0071">BBa_C0071</a>) (pSB6A1), Prhl(LR)(<a href="http://parts.igem.org/Part:BBa_K1529310">BBa_K1529310</a>) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span>(pSB3K3) (Fig. 3-2-3-5-1)<br><br>
 				B. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span>(<a href="http://parts.igem.org/Part:BBa_C0171">BBa_C0171</a>) (pSB6A1), Prhl(LR) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-5-2)<br>
 				C. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(RL)(<a href="http://parts.igem.org/Part:BBa_K1529300">BBa_K1529300</a>) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-5-3)<br>
 				D. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> (pSB6A1), Prhl(RL) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-5-4)<br>
 				E. pSB6A1, pSB3K3 …Nagative control (Fig. 3-2-3-5-5)<br><br>
 
				&#8545;. Improvment the wild type Prhl <br>
				 A. Prhl(<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-5-6)<br><br>
 				 B. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <i>LVA</i> (pSB6A1), Prhl(NM) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span>(<a href="http://parts.igem.org/Part:BBa_K1949060">BBa_ K1949060</a>) (pSB3K3) (Fig. 3-2-3-5-7)<br>
				 C. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(M) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-5-8)<br>
				 D. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(WT) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-5-9)<br>
				 E. pSB6A1, pSB3K3 …Nagative control (Fig. 3-2-3-5-5)<br><br>
 
				&#8546;. Comparison the improved Prhl by iGEM 2014 Tokyo_Tech Team to our original improved Prhl<br>
 				 A. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(LR) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp </span>(pSB3K3) (Fig. 3-2-3-5-1)<br>
				 B. Pcon - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">rhlR</span> - <span style ="font-style : italic">LVA</span> (pSB6A1), Prhl(NM) - <span style="font-style : italic">rbs</span> - <span style="font-style : italic">gfp</span> (pSB3K3) (Fig. 3-2-3-5-7)<br>
				 C. pSB6A1, pSB3K3…Negative control (Fig. 3-2-3-5-5)<br><br>

                -Medium<br>
				 LB medium AK:<br>
				    LB medium containing ampicillin (50 microg/ mL) and kanamycin (50 microg/mL)<br>
				 LB medium K:<br>
				    LB medium containing kanamycin (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>

				I. Improved Prhl by iGEM 2014 Tokyo_Tech Team and <span style="font-style : italic">rhlR</span> assay <br><br>

				1) Prepare overnight culture for each sample in 3mL LB medium AK with vigorous shaking.<br><br>

				2) Dilute the overnight cultures to 1 / 60 in fresh LB medium AK (1.2 mL).<br><br>

				3) Incubate the fresh cultures for 1 h with vigorous shaking. <br><br>

				4) Add C4 or DMSO to each 500 microL sample at the final concentration 10 microM or 100 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 a measurement wavelength.  <br><br>  

				8) Measure the turbidity at 600 nm.<br><br>


				II. Improvement the wild type Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>)<br><br>

				1) Introduce a point mutation to -10 region of Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>)-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3) by inverse PCR using wild type Prhl(<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>)-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3) as a template and divergent primers. The sequences of the six forward premiers and of one reverse primer are shown below.<br><br>

				WT 　  5'-tcctgtgaaatctggcagttaccgttagctttcgaatt ggctaaaaagtgttctactagtagcg-3'<br>
				F-Mu1  5'-tcctgtgaaatctggcagttaccgttagctttcgaatt ggntaaaaagtgttctactagtagcg-3' <br>
				F-Mu2  5'-tcctgtgaaatctggcagttaccgttagctttcgaatt ggcnaaaaagtgttctactagtagcg-3' <br>
				F-Mu3  5'-tcctgtgaaatctggcagttaccgttagctttcgaatt ggctnaaaagtgttctactagtagcg-3' <br>
				F-Mu4  5'-tcctgtgaaatctggcagttaccgttagctttcgaatt ggctanaaagtgttctactagtagcg-3' <br>
				F-Mu5  5'-tcctgtgaaatctggcagttaccgttagctttcgaatt ggctaanaagtgttctactagtagcg-3' <br>
				F-Mu6  5'-tcctgtgaaatctggcagttaccgttagctttcgaatt ggctaaanagtgttctactagtagcg-3' <br>
				R       5'-aattcgaaagctaacggtaactgcca-3' (5' phosphorylated)<br><br>

				2) Prepare strains containing the pMu1~6 plasmids containing mutated Prhl-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">gfp</span> (pSB3K3).<br><br>

				3) Streak the transformants on an agar plate containing kanamycin (50 microg / mL) and incubate overnight.<br><br>

				4) Pore the LB medium K at the extent of covering the surface of the agar, and collect the colonies with a spreader. Then, incubate them in 5 mL LB medium K for 16 h with vigorous shaking.<br><br>

				5) Extract the plasmid, and introduce Prhl(mut)-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB3K3) and Pcon-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">rhlR</span>-<span style ="font-style : italic">LVA</span> to XL-1 Blue.<br><br>

				6) Pore 200 microL LB medium AK containing C4 (6 microM) in one well of a 96-well plate and inoculate one colony to the well. Incubate the plate for 16 h. <br><br>

				7) Transfer 100 microL of the culture from the above samples to another plate that is suitable for fluorescence measurement. Measure the GFP RFU of GFP / Turbidity at 490 nm as an exciting wavelength and 525 nm as an emission wavelength as well as the turbidity at 600 nm.<br><br>

				8) Select mutants that have higher SN ratio than wild type Prhl (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) and grow them in 3mL LB medium AK overnight with vigorous shaking. <br><br>

				9) Dilute the overnight cultures to 1 / 60 in fresh LB medium AK (1.2 mL).<br><br>

				10) Incubate the fresh cultures for 1 h with vigorous shaking. <br><br>

				11) Add C4, C12 or DMSO to each 500 microL sample at the final concentration 10 microM. <br><br>

				12) Incubate the samples for 4 h with vigorous shaking.<br><br>

				13) Add 100 microL of the samples to each well of a plate reader.<br><br>

				14) Measure RFU of GFP at 490 nm as an exciting wavelength, 525 nm as a measurement wavelength.    <br><br>

				15) Measure the turbidity at 600 nm.<br><br><br>



				III. Comparison the improved Prhl promoter by iGEM 2014 Tokyo_Tech Team to our original improved Prhl promoter<br><br>

				1) Prepare overnight cultures for each sample in 3mL LB medium AK with vigorous shaking.<br><br>

				2) Dilute the overnight cultures to 1 / 60 in fresh LB medium AK (1.2 mL).<br><br>

				3) Incubate the fresh cultures for 1 h with vigorous shaking.<br><br>

				4) Add C4, 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 a measurement 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">(1) Gerardo Medina et al. (2003) Mechanism of Pseudomonas aeruginosa RhlR Transcriptional Regulation of the rhlAB Promoter.<br>
				BACTERIOLOGY 185: 5976-5983<br><br>

				(2) John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. 

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


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