View source for Team:Tokyo Tech/Toxin Assay/Adjustment of Expression of MazF

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			<h1 align="center">3-1-1 Adjustment of MazF Expression<br><br></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 experiment</a></h3>
				<h3 class="link"><a href="#results">3. Results & discussion</a></h3>
				<h3 class="link"><a href="#methods">4. Materials and methods</a></h3>
				<h3 class="link"><a href="#construction"><font size="2.7">&nbsp;&nbsp;&nbsp;4-1. Construction</font></a></h3>
				<h3 class="link"><a href="#protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;4-2. Assay protocol</font></a></h3>
				<h3 class="link"><a href="#reference">5. Reference</a></h3>
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				<h2><span>1. Introduction</span></h2>
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				<p class="normal_text">In order to control cell growth as we desire using the <span style ="font-style : italic">mazEF</span> system, it is necessary to adjust the expression level of <span style ="font-style : italic">mazF</span>. It has been reported that MazF has very strong ability to inhibit cell growth and that <span style ="font-style : italic">mazE</span> expression can not recover from it when <span style ="font-style : italic">mazF</span> is expressed at high level[1]. Therefore, we here explored the relationship between concentration of the expression inducer for <span style ="font-style : italic">mazF</span> (arabinose in this experiment) and expression level of it; such information is important for operating our final genetic circuits properly. 


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				<h2><span>2. Summary of the experiment</span></h2>
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				<p class="normal_text">A pSB6A1-based plasmid containing both the PBAD &#8208; <span style ="font-style : italic">rbs</span> &#8208; <span style ="font-style : italic">mazF</span> and the Pcon &#8208; <span style ="font-style : italic">rbs</span> &#8208; <span style ="font-style : italic">gfp</span> cassettes was constructed. For control experiments, a pSB6A1-based plasmid containing only the Pcon - <span style ="font-style : italic">rbs</span> &#8208; <span style ="font-style : italic">gfp</span> cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac &#8208; <span style ="font-style : italic">rbs</span> cassette was constructed. These plasmids were co-introduced into <span style ="font-style : italic">E. coli</span> cells of which growth was controlled by the expression of <span style ="font-style : italic">mazF</span>.<br>
                        <p class="normal_text">The plasmid shown in Fig. 3-1-1-2-2 (PBAD &#8208; <span style ="font-style : italic">rbs</span> &#8208; <span style ="font-style : italic">mazF</span> and the Pcon &#8208; <span style ="font-style : italic">rbs</span> &#8208; <span style ="font-style : italic">gfp</span>) has been registered as Best Composite Part (<a href="http://parts.igem.org/Part:BBa_K1949102">BBa_K1949102</a>).<br><br>
                        <p class="normal_text">Transformants shown below were prepared.<br>

                                <h3 id="fig.3-1-1-2-1"> </h3>                                
                                <p class="normal_text"><span style ="font-style : italic">E. coli</span> A: carrying the Pcon &#8208; <span style ="font-style : italic">rbs &#8208; gfp</span> (pSB6A1) , Plac &#8208; <span style ="font-style : italic">rbs</span> (pSB3K3)</p>
　　　　　　　　　　　　　　　　<div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f2/T--Tokyo_Tech--3-1-2-2-5-2.png" height="150"><br></div>
                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-2-1 </span> Plasmid diagram of <span style ="font-style : italic">E. coli</span> A</span>
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                                <h3 id="fig.3-1-1-2-2"> </h3>
                                <p class="normal_text"><span style ="font-style : italic">E. coli</span> B: carrying the PBAD &#8208; <span style ="font-style : italic">rbs &#8208; mazF &#8208; tt</span> &#8208; Pcon &#8208; <span style ="font-style : italic">rbs &#8208; gfp</span> (pSB6A1) , Plac &#8208; <span style ="font-style : italic">rbs</span> (pSB3K3)</p>

                                <h3 id="fig.3-1-2-1-3-2"> </h3>　　　　　　　　　　　　　　　　
                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/c/ca/T--Tokyo_Tech--3-1-2-2-5-5.png" height="250"><br></div>
				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-2-2 </span> Plasmid diagram of <span style ="font-style : italic">E. coli</span> B</span>
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                                <p class="normal_text">To express <span style ="font-style : italic">mazF</span>, arabinose was added so that the final concentration became 0.2, 0.02, 0.002, 0.0002, and 0%. Samples were incubated with vigorous shaking for 24 h at 37℃, and the turbidity and the Relative Fluorescence Units (RFU) of GFP were measured.</p>
				
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		<div id="results" class="container">
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				<h2><span>3. Results & discussion</span></h2>
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				<p class="normal_text">It was found that cell growth of <span style ="font-style : italic">E. coli</span> B was inhibited under arabinose (inducer for <span style ="font-style : italic">mazF</span>) concentrations of over 0.02% (<a href="#fig.3-1-1-3-1">Fig.3-1-1-3-1</a>). Interestingly, at arabinose concentration of 0.2%, RFU of GFP fell markedly, regardless of <span style ="font-style : italic">mazF</span> expression, suggesting that high arabinose concentrations may inhibit <span style ="font-style : italic">gfp</span> expression or prevent GFP from exerting fluorescence. Taken together, it is concluded that the concentration of arabinose should be 0.02%. 
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                                <h3 id="fig.3-1-1-3-1"> </h3>
				<div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6b/T--Tokyo_Tech--Description_Toxin1.png" width="400px"/></div>
				

				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-1-3-1 </span> Relative value of turbidity, RFU of GFP and RFU of GFP / turbidity. <a href="#fig.3-1-1-2-1"><span style ="font-style : italic">E. coli</span> A</a> and <a href="#fig.3-1-1-2-2"><span style ="font-style : italic">E. coli</span> B</a> were cultured in the presence of indicated amounts of arabinose, and  turbidity (upper graph), RFU of GFP (middle graph), and RFU / turbidity (lower graph) were measured. Also, the same cells  were cultured in the absence of arabinose, and measurements were performed similarly as above. The ratio of the former values to the latter values were calculated.
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				<h2><span>4. Materials and methods</span></h2>
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					<h3><span>4-1. Construction</span></h3>
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				<p class="normal_text"> -Strain<br>
				 　All the samples were XL1-Blue strain.</p><br>

				<p class="normal_text">-Plasmids<br>
				&nbsp;&nbsp;&nbsp;&nbsp;<a href="#fig.3-1-1-2-1"><span style ="font-style : italic">E. coli</span> A</a>: Pcon &#8208; <span style ="font-style : italic">rbs</span> &#8208; <span style ="font-style : italic">gfp</span> (pSB6A1) + Plac &#8208; <span style ="font-style : italic">rbs</span> (pSB3K3)<br>

				&nbsp;&nbsp;&nbsp;&nbsp;<a href="#fig.3-1-1-2-2"><span style ="font-style : italic">E. coli</span> B</a>: PBAD &#8208; <span style ="font-style : italic">rbs</span> &#8208; <span style="font-style : italic">mazF</span> &#8208; <span style ="font-style : italic">tt</span> &#8208; Pcon &#8208; <span style ="font-style : italic">rbs</span> &#8208; <span style="font-style : italic">gfp</span> (pSB6A1) + Plac &#8208; <span style ="font-style : italic">rbs</span> (pSB3K3)</p><br>
                                <p class="normal_text">PBAD (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) , Plac (<a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a>) , Pcon (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>) , <span style ="font-style : italic">gfp</span> (<a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a>) ,<br>
                                &nbsp;&nbsp;&nbsp;&nbsp;<span style ="font-style : italic">rbs</span> (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) , <span style ="font-style : italic">mazF</span> (<a href="http://parts.igem.org/Part:BBa_K1096002">BBa_K1096002</a>) , <span style ="font-style : italic">tt</span> (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a>)

                                


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					<h3><span>4-2. Assay protocol</span></h3>
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					<p class="normal_text">Pre-culture<br><br>
					  1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).<br><br>
					  2. Incubate with vigorous shaking for 12 h.<br><br><br>
　　 					<p class="normal_text">Incubation and Assay<br><br>
					  1. Measure the turbidity of the pre-cultures.<br><br>
					  2. Dilute the pre-cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.<br><br>
					  3. Incubate with vigorous shaking so that the turbidity becomes 0.03.<br><br>
					  4. Add arabinose so that the final concentration becomes 0.2%, 0.02%, 0.002% 0.0002% and 0%.<br><br>
				  	  5. Incubate with vigorous shaking for 24 h, and measure the turbidity and the RFU of GFP.


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				<h2><span>5. Reference</span></h2>
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                                <h3 id="reference3-1-1-6-1"> </h3>
				<p class="normal_text">[1] Hazan, R., B. Sat, and H. Engelberg-Kulka. <span style ="font-style : italic">E. coli</span> <span style ="font-style : italic">mazEF</span> mediated cell death is triggered by various stressful conditions. J. Bacteriol. 2004 Dec;186(24):8295-8300.
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