Difference between revisions of "Team:Tokyo Tech/Experiment/Realizing the Payoff Matrix"

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      <h1>Realizing the Payoff Matrix</h1>
 
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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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          <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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      <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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      <h4 align="center" class="fig"><strong>Fig.&nbsp;3-2-1-1.</strong>&nbsp;C4HSL-dependent CmR expression</h4>
 
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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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      <h4 align="center" class="fig"><strong>Fig.&nbsp;3-2-2-1.</strong>&nbsp;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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                <h3 id="Summary1" 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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      <h4 align="center" class="fig"><strong>Fig.&nbsp;3-2-2-2.</strong>&nbsp;Cells for the experiment to realize the payoff matrix. of Prisoner B coli</h4>
 
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          <h2 id="Results" class="smalltitle">3. Results</h2>
 
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              <h3 id="Result1" class="sub5">3.1. Arabinose dependent FimE expression</h3>
 
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　　　　　　    <p class="text2">私たちは、4種類のarabinose濃度でFimBが働くかどうかを、GFPを用いたレポーターアッセイによって確かめた。
 
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　Figure(図番号) は、default ONのサンプルが、arabinose誘導によって、OFF状態に切り替わった結果を示している。
 
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またFigure(図番号)は、default OFFのサンプルが、arabinose誘導によって、ON状態に切り替わった結果を示している。
 
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Figure(図番号) shows our experimental results of FimB and Fimswitch. From the results of the reporter cell C and D, inversion from ON to OFF and OFF to ON by endogenous proteins are negligible. レポーターセルE,Fの結果から、FImEの発現はヒストグラムの波形にほとんど影響を与えないことがわかる。
 
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以上の2つの結果から、FimBが理想的に両反転を起こしていることがわかる。
 
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                  <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/1/1a/Tokyo_Tech_arabinose_fimB_result1.png" width="800px"/>
 
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      <td width="980px">
 
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      <h4 align="center" class="fig"><strong>Fig. 3-4-3-1.</strong></h4>
 
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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>
 
−
              <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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                  <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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      <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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　　　　　　    <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>
 
−
      <td>
 
−
      </tr>
 
−
      </table>
 
−
　　　　　　    <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>
 
−
      <td>
 
−
      </tr>
 
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      </table>
 
−
　　　　　　    <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>
 
−
      <td>
 
−
      </tr>
 
−
      </table>
 
−
　　　　　　    <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>
 
−
5. Add 30 microL of suspension in the following medium.<br>
 
−
&nbsp;&nbsp;&nbsp;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>
 
−
&nbsp;&nbsp;&nbsp;②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)<br>
 
−
6. Grow the samples of cells at 37°C for more than 8 hours.<br>
 
−
7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)<br><br>
 
−
 
−
              <h3 id="Protol2" class="sub6">4.2.2. C4HSL-dependent CmR expression assay (With an ssrA tag)</h3>
 
−
                    <p class="text4">
 
−
<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>
 
−
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>
 
−
<br>
 
−
<strong>-Procedure</strong><br>
 
−
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>
 
−
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>
 
−
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
 
−
4. Suspend the pellet in 1mL of LB containing Amp and Kan.<br>
 
−
5. Add 30 microL of suspension in the following medium.<br>
 
−
&nbsp;&nbsp;&nbsp;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>
 
−
&nbsp;&nbsp;&nbsp;②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)<br>
 
−
6. Grow the samples of cells at 37°C for more than 8 hours.<br>
 
−
7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)<br></p>
 
−
              <h3 id="Protol3" class="sub6">4.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag</h3>
 
−
                    <p class="text4">
 
−
<strong>-samples</strong><br>
 
−
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#1<br>
 
−
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>
 
−
<br><strong>-Procedure</strong><br>
 
−
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>
 
−
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>
 
−
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.<br>
 
−
4. Suspend the pellet in 1 mL of LB containing Amp and Kan.<br>
 
−
5. Add 30 microL of suspension in the following medium.<br>
 
−
&nbsp;&nbsp;&nbsp;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>
 
−
&nbsp;&nbsp;&nbsp;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>
 
−
&nbsp;&nbsp;&nbsp;③) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)<br>
 
−
6. Grow the samples of cells at 37°C for more than 8 hours.<br>
 
−
7. Measure the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)<br>
 
−
              <h3 id="Protol4" class="sub6">4.2.4. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)</h3>
 
−
                    <p class="text4">
 
−
<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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&nbsp;&nbsp;&nbsp;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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&nbsp;&nbsp;&nbsp;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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Latest revision as of 12:45, 18 September 2015