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| − | {{Team:Tokyo Tech/template2015}}
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| − | <meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
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| − | <meta name="generator" content="MediaWiki 1.16.5" />
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| − | </style>
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| − | </head>
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| − | <body>
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| − | <div id="titlearea">
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| − | <h1>Realizing the Payoff Matrix</h1>
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| − | </div>
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| − | <div id="titlebottom">
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| − | </div>
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| − | <div class="texttop">
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| − | <img src="https://static.igem.org/mediawiki/2015/a/a4/Tokyo_Tech_textarea_top.png">
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| − | </div>
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| − | <div class="textarea">
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| − | <h2 class="content2">contents</h2>
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| − | <h3 class="link"><a href="#Introduction">1. Introduction</a></h3>
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| − | <h3 class="link"><a href="#Summary">2. Summary of the Experiment</a></h3>
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| − | <h3 class="link2"><a href="#Summary1">2.1. Realizing the Payoff Matrix of Prisoner A <i>coli</i></a></h3>
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| − | <h3 class="link2"><a href="#Summary2">2.2. Realizing the Payoff Matrix of Prisoner B <i>coli</i></a></h3>
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| − | <h3 class="link"><a href="#Results">3. Results</a></h3>
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| − | <h3 class="link2"><a href="#Result1">3.1. Realizing the Payoff Matrix of Prisoner A <i>coli</i></a></h3>
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| − | <h3 class="link2"><a href="#Result2">3.2. Realizing the Payoff Matrix of Prisoner B <i>coli</i></a></h3>
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| − | <h3 class="link"><a href="#Materials">4. Materials and Methods</a></h3>
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| − | <h3 class="link2"><a href="#Const">4.1. Construction</a></h3>
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| − | <h3 class="link2"><a href="#Protocol">4.2. Assay Protocol</a></h3>
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| − | <h3 class="link3"><a href="#Protol1">4.2.1. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)</a></h3>
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| − | <h3 class="link3"><a href="#Protol2">4.2.2. 3OC12HSL concentration-dependent CmR expression assay</a></h3>
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| − | <h3 class="link3"><a href="#Protol3">4.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag</a></h3>
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| − | <h3 class="link3"><a href="#Protol4">4.2.4. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)</a></h3>
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| − | <h3 class="link"><a href="#Reference">5. Reference</a></h3>
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| − | <br>
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| − | </div>
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| − | <div class="textbottom">
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| − | <img src="https://static.igem.org/mediawiki/2015/9/97/Tokyo_Tech_textarea_bottom.png">
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| − | </div>
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| − | <div class="texttop">
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| − | <img src="https://static.igem.org/mediawiki/2015/a/a4/Tokyo_Tech_textarea_top.png">
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| − | </div>
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| − | <div class="textarea">
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| − | <h2 id="Introduction" class="smalltitle">1. Introduction</h2>
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| − | <p class="text">We genetically engineered two prisoner <i>coli</i>, Prisoner A and Prisoner B. They are able to cooperate or to defect. The genetic circuits, with the improved chloramphenicol resistant protein(CmR) part, of Prisoner A and B are shown in Fig. 3-2-1-1. Our goal in this project is to realize the payoff matrix (Fig. 3-2-1-2) with the four types of growth inhibition. Using the improved plasmids we constructed, our <i>E.coli</i> version payoff matrix is realized through wet experiments. </p>
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| − | <table width="940 px" border="0px">
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| − | <tr>
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| − | <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/9/93/Tokyo_Tech_realizing_summary1.png" width="600px"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="940px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-2-1-1.</strong> C4HSL-dependent CmR expression</h4>
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| − | <td>
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| − | </tr>
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| − | </table><br>
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| − | <h2 id="Summary" class="smalltitle">2. Summary of the Experiment</h2>
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| − | <h3 id="Summary1" class="sub5">2.1. Realizing the Payoff Matrix of Prisoner A <i>coli</i></h3>
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| − | <p class="text2">The cell growth of the Prisoner <i>colis</i> containing the pairs of plasmids (1) and (2) in the lower chloramphenicol (Cm) concentration (75 microg/mL) were measured and the payoff matrix was realized.</p>
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| − | <table width="940 px" border="0px">
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| − | <tr>
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| − | <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/3/3b/Tokyo_Tech_realizing3.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="940px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-2-2-1.</strong> Cells for the experiment to realize the payoff matrix of Prisoner A <i>coli</i></h4>
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| − | <td>
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| − | </tr>
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| − | </table><br>
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| − | <h3 id="Summary2" class="sub5">2.2. Realizing the Payoff Matrix of Prisoner B <i>coli</i></h3>
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| − | <p class="text2">We have prepared a new pair of plasmids, (3) and (4), as in the following (Fig. 3-2-2-2.). The cell growth of the Prisoner <i>colis</i> containing the pairs of plasmids (3) and (4) in the lower chloramphenicol (Cm) concentration (75 microg/mL) and also in the lower 3OC12HSL concentration were measured and the payoff matrix was realized.</p>
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| − | <table width="940 px" border="0px">
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| − | <tr>
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| − | <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a2/Tokyo_Tech_realizing4.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="940px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-2-2-2.</strong> Cells for the experiment to realize the payoff matrix of Prisoner B <i>coli</i></h4>
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| − | <td>
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| − | </tr>
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| − | </table><br>
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| − | <p class="text2">Using the pair of plasmids we constructed, our E.<i>coli</i> version payoff matrix with the four types of growth inhibition was replicated through wet experiments. The cell growth of the Prisoner <i>coli</i>s containing the pairs of plasmids (3) and (4), grown under the lower Cm concentration and also with the lower 3OC12HSL concentration, were observed. (refer protocol 5-2-3.) Results are shown here (link to 3.3.1.).
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| − | <h2 id="Results" class="smalltitle">3. Results</h2>
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| − | <h3 id="Result1" class="sub5">3.1. Realizing the Payoff Matrix of Prisoner A <i>coli</i></h3>
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| − | <h3 id="Result2" class="sub5">3.2. FLA analysis</h3>
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| − | <p class="text2">写真とシークエンスデータ</p>
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| − | <h2 id="Materials" class="smalltitle">4. Materials and Methods</h2>
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| − | <h3 id="Const" class="sub5">4.1. Construction</h3>
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| − | <h3 class="sub5">-Strain</h3>
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| − | <p class="text2">All the samples were JM2.300 strain.</p>
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| − | <h3 class="sub5">-Plasmids</h3>
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| − | <p class="text2">(1) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)</p>
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| − | <table width="980 px" border="0px">
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| − | <tr>
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| − | <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/b/be/Tokyo_Tech_Pcon_rhlR_TT_Plux_CmRssrA_Plac_lasI.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="980px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-1-4-1.</strong></h4>
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| − | <td>
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| − | </tr>
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| − | </table>
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| − | <p class="text2">(2) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +promoter less_lasI (pSB3K3)</p>
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| − | <table width="980 px" border="0px">
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| − | <tr>
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| − | <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/2/24/Tokyo_Tech_Pcon_rhlR_TT_Plux_CmRssrA_lasI.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="980px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-1-4-2.</strong></h4>
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| − | <td>
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| − | </tr>
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| − | </table>
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| − | <p class="text2">(3) Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)</p>
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| − | <table width="980 px" border="0px">
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| − | <tr>
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| − | <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/e/e0/Tokyo_Tech_Pcon_rhlR_TT_CmR_Plac_lasI.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="980px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-1-4-3.</strong></h4>
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| − | <td>
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| − | </tr>
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| − | </table>
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| − | <p class="text2">(4) Pcon_rhlR_TT_Plux_CmR (pSB6A1) +promoter less_lasI (pSB3K3)</p>
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| − | <table width="980 px" border="0px">
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| − | <tr>
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| − | <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/3/30/Tokyo_Tech_Pcon_rhlR_TT_Plux_CmR_lasI.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="980px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-1-4-4.</strong></h4>
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| − | <td>
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| − | </tr>
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| − | </table>
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| − | <p class="text2">(5) Negative control1: Pcon_rhlR_TT_promoter less_CmR (pSB6A1) + Plac_lasI (pSB3K3)</p>
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| − | <table width="980 px" border="0px">
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| − | <tr>
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| − | <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/e/eb/Tokyo_Tech_Pcon_rhlR_TT_CmR_Plac_lasI.2png.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="980px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-1-4-5.</strong></h4>
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| − | <td>
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| − | </tr>
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| − | </table>
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| − | <p class="text2">(6) Negative cotrol2:Pcon_rhlR_TT_promoter less_CmR (pSB6A1) +promoter less_lasI (pSB3K3)</p>
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| − | <table width="980 px" border="0px">
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| − | <tr>
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| − | <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/4/45/Tokyo_Tech_Pcon_rhlR_TT_CmR_lasI.png"/>
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| − | </td>
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| − | </tr>
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| − | <tr>
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| − | <td width="980px">
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| − | <h4 align="center" class="fig"><strong>Fig. 3-1-4-6.</strong></h4>
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| − | <td>
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| − | </tr>
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| − | </table>
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| − | <h3 id="Protocol" class="sub5">4.2. Assay Protocol</h3>
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| − | <h3 id="Protol1" class="sub6">4.2.1. C4HSL-dependent CmR expression assay</h3>
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| − | <p class="text4">
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| − | <strong>-samples</strong><br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋ promoter less_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1) ＋ promoter less_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#2<br>
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| − | </p>
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| − | <p class="text4"><br>
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| − | <strong>-Procedure</strong><br>
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| − | 1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.<br>
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| − | 2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.<br>
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| − | 3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
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| − | 4. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
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| − | 5. Add 30 microL of suspension in the following medium.<br>
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| − | ①)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)<br>
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| − | ②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)<br>
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| − | 6. Grow the samples of cells at 37°C for more than 8 hours.<br>
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| − | 7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)<br><br>
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| − |
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| − | <h3 id="Protol2" class="sub6">4.2.2. C4HSL-dependent CmR expression assay (With an ssrA tag)</h3>
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| − | <p class="text4">
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| − | <strong>-samples</strong><br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#2<br>
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| − | <br>
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| − | <strong>-Procedure</strong><br>
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| − | 1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.<br>
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| − | 2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.<br>
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| − | 3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
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| − | 4. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
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| − | 5. Add 30 microL of suspension in the following medium.<br>
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| − | ①)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)<br>
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| − | ②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)<br>
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| − | 6. Grow the samples of cells at 37°C for more than 8 hours.<br>
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| − | 7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)<br></p>
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| − | <h3 id="Protol3" class="sub6">4.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag</h3>
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| − | <p class="text4">
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| − | <strong>-samples</strong><br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#2<br>
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| − | <br><strong>-Procedure</strong><br>
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| − | 1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.<br>
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| − | 2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.<br>
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| − | 3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
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| − | 4. Suspend the pellet in 1 mL of LB containing Amp and Kan.<br>
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| − | 5. Add 30 microL of suspension in the following medium.<br>
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| − | ①) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (6 microL of 25 microg/mL) + 99.5% ethanol (6 microL)<br>
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| − | ②) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (9 microL of 25 microg/mL) + 99.5% ethanol (3 microL)<br>
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| − | ③) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)<br>
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| − | 6. Grow the samples of cells at 37°C for more than 8 hours.<br>
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| − | 7. Measure the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)<br>
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| − | <h3 id="Protol4" class="sub6">4.2.4. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)</h3>
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| − | <p class="text4">
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| − | <strong>-Samples</strong><br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#2<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#1<br>
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| − | Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#2<br>
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| − | <br><strong>-Procedure</strong><br>
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| − | 1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.<br>
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| − | 2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.<br>
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| − | 3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
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| − | 4. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
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| − | 5. Add 30 microL of suspension in the following medium.<br>
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| − | ①) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (75 microg/mL)<br>
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| − | ②) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (75 microg/mL)<br>
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| − | 6. Grow the samples of cells at 37°C for more than 8 hours.<br>
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| − | 7. Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)<br>
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| − | <h2 id="Reference" class="smalltitle">5. Reference</h2>
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| − | <p class="text">1. Bo Hu <em>et al.</em> (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619</p>
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