Team:Tokyo Tech/Project

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      <h2 class="title">Project</h2>
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             <span class="meta">Learning economics with Bank <i>E. coli</i> </span>
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             <span class="meta">Learning economics by Bank <em>E. coli</em> </span>
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                       <p align="center" class="title-small">Content</p>
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                       <p align="center" class="title-small">Contents</p>
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              <p align="left" class="info-24"><a href="#1">1. Project planning</a></p>    
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              <p align="left" class="info-24"><a href="#1">1. Project Planning: Interaction with General Public</a></p>    
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              <p align="left" class="info-24"><a href="#2">2. Interdependence between Company and Customer</a></p>
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              <p align="left" class="info-24"><a href="#2">2. Mutualism between Company and Customer</a></p>
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                       <p align="left" class="info-18"><a href="#2.1">2.1 Molecular Basis of Interdependence</a></p>
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                       <p align="left" class="info-18"><a href="#2.1">2.1 Molecular Basis of Mutualism</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.1.1">2.1.1 Company</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.1.1">2.1.1 Company</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.1.2">2.1.2 Customer</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.1.2">2.1.2 Customer</a></p>
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                       <p align="left" class="info-18"><a href="#2.2">2.2. Native Prhl Promoter does not satisfy  
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                       <p align="left" class="info-18"><a href="#2.2">2.2 Native Prhl promoter does not satisfy  
requirement from system analysis</a></p>
requirement from system analysis</a></p>
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                       <p align="left" class="info-18"><a href="#2.3">2.3. The improvement of Prhl promoter</a></p>
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                       <p align="left" class="info-18"><a href="#2.3">2.3 The improvement of Prhl promoter</a></p>
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                       <p align="left" class="info-18"><a href="#2.4">2.4. HSL-dependent responses of Company <i>E. coli</i> with improved promoter and Customer <i>E. coli</i></a></p>
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                       <p align="left" class="info-18"><a href="#2.4">2.4 AHL-dependent responses of Company <em>E. coli</em> with improved promoter and Customer <em>E. coli</em></a></p>
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                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.4.1">2.4.1 Company</a></p>
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                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.4.1">2.4.1 Company with improved promoter</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.4.2">2.4.2 Customer</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#2.4.2">2.4.2 Customer</a></p>
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                       <p align="left" class="info-18"><a href="#2.5">2.5. Assay of symbiosis between Company and Customer by co-culture</a></p>
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                       <p align="left" class="info-18"><a href="#2.5">2.5 Assay of symbiosis between Company and Customer by co-culture</a></p>
      <p align="left" class="info-24"><a href="#3">3. Addition of Bank</a></p>
      <p align="left" class="info-24"><a href="#3">3. Addition of Bank</a></p>
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                       <p align="left" class="info-18"><a href="#3.1">3.1. Motivation</a></p>
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                       <p align="left" class="info-18"><a href="#3.1">3.1 Motivation</a></p>
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                       <p align="left" class="info-18"><a href="#3.2">3.2. Genetic circuit design of Bank <i>E. coli</i></a></p>
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                       <p align="left" class="info-18"><a href="#3.2">3.2 Genetic circuit design of Bank <em>E. coli</em></a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.1">3.2.1 Distribution state</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.1">3.2.1 Distribution state</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.2">3.2.2 Change to Collection State </a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.2">3.2.2 Change to Collection State </a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.3">3.2.3 Collection State</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.3">3.2.3 Collection State</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.4">3.2.4 Change to distribution state</a></p>
                       <p align="left" class="info" style="text-indent:40px;"><a href="#3.2.4">3.2.4 Change to distribution state</a></p>
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                       <p align="left" class="info-18"><a href="#3.3">3.3 Modeling:Bank actually helps economy development</a></p>
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                       <p align="left" class="info-18"><a href="#3.3">3.3 Modeling:Bank actually helps the development of economy</a></p>
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                       <p align="left" class="info-24"><a href="#4">4. Economic Wave</a></p>
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                       <p align="left" class="info-24"><a href="#4">4. Economic Wave: Advice from Entrepreneurs</a></p>
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      <p align="left" class="info-24"><a href="#5">5. Reference</a></p>
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      <p align="left" class="info-24"><a href="#5">5. Project Evaluation</a></p>
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      <p align="left" class="info-24"><a href="#6">6. Reference</a></p>
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                   <td colspan="2"><h2>1.Project planning</h2></td>
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                   <td colspan="2"><h2>1.Project Planning: Interaction with General Public</h2></td>
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                   <td colspan="2"><div align="center">Fig. 2-1-1. Our  members discussing about the project with the visitor. </div></td>
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                   <td colspan="2"><div align="center"><strong>Fig. 2-1-1.</strong> Our  members discussing about the project with the visitor </div></td>
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                   <td colspan="2"><p class="info-18">This year, we decided to make an educational tool for economics by using <i>E. coli</i>. This tool, which is supported with BioBrick parts and modeling, can be easily adapted to not only iGEMers, but also other biology student who are great human resource of innovation. </p></td>
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                   <td colspan="2"><p class="info-18">This year, we decided to make an educational tool for economics by using <em>E. coli</em>. This tool, which is supported with BioBrick parts and modeling, can be easily adapted to not only iGEMers, but also other students majoring in biology who are great human resource of innovation. </p></td>
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                   <td height="9" colspan="2"><p class="info-18">At the project planning stage of this year, we took part in a poster session held at the school festival of the University of Tokyo. We showed our preliminary plans, such as <em>E. coli</em>solar battery, and fertilizer. (See Policy and Practice for more details) (Fig. 2-1-1). After communicating with people engaged in business, we realized that we do not know much about economic system. This lack of knowledge, which may be shared with other iGEMers, can be an obstacle for innovation from our research activity. In order to solve this, we thought of making an educational tool for economics by using <em><i>E. coli</i></em> and BioBrick, which are familiar for iGEMers.</p></td>
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                   <td height="9" colspan="2"><p class="info-18">At the project planning stage of this year, we took part in a poster session held at the school festival of the University of Tokyo. We showed our preliminary plans, such as <em>E. coli</em> solar battery, and fertilizer. (See <a href="https://2014.igem.org/Team:Tokyo_Tech/Policy_and_Practices#Poster">Policy and Practice</a> for more details) (Fig. 2-1-1). After communicating with people engaged in business, we realized that we do not know much about economic system. This lack of knowledge, which may be common with other iGEMers, can be an obstacle for innovation from our research activity. In order to solve this, we thought of making an educational tool for economics by using <em>E. coli</em> and BioBrick, which are familiar for iGEMers.</p></td>
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                   <td height="9" colspan="2"><div align="center">Fig. 2-1-2. Relationship  among three types of <i>E. coli</i></div></td>
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                   <td height="9" colspan="2"><div align="center"><strong>Fig. 2-1-2.</strong> Relationship  among three types of <em>E. coli</em></div></td>
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                   <td height="9" colspan="2"><p class="info-18">Our educational tool has three types of <em><i>E. coli</i></em>. They are Bank, Company, and Customer (Fig. 2-1-1). Like the exchange of money and products in the real economy, we designed these three <em><i>E. coli</i></em> to exchange Product and Money in the tool’s economic system. The Company makes Product, and sells them to the Customer. On the other hand, the Customer pays Money to buy the Product made by the Company. With this mutual action, the Customer and the Company build mutualistic relationship. Additionally, the Bank regulates the Money supply in the market. It functions as a central bank like FRB and European Central Bank (ECB) (see here for more information about central bank). Here, Product and Money were represented by 3OC12HSL and C4HSL, the signaling molecules of the quorum sensing, respectively. </p></td>
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                   <td height="9" colspan="2"><p class="info-18">Our educational tool has three types of <em>E. coli</em>. They are Bank, Company, and Customer (Fig. 2-1-2).Like the exchange of money and products in the real economy, we designed these three <em>E. coli</em> to exchange Product and Money in the tool’s economic system. The Company makes Product, and sells them to the Customer. On the other hand, the Customer pays Money to buy the Product made by the Company. With this mutual action, the Customer and the Company build mutualistic relationship. Additionally, the Bank regulates the Money supply in the market. It functions as a central bank like FRB and European Central Bank (ECB) (see <a href="http://en.wikipedia.org/wiki/Central_bank">here</a> for more information about central bank). Here, Product and Money were represented by 3OC12HSL and C4HSL, the signaling molecules of the quorum sensing, respectively.</p></td>
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                  <td colspan="2"><p class="info-18">With this mutual action, Customer and Company build  interdependent relationship. Additionally, Bank regulates the Money supply in  the market. It functions as a central bank like FRB and European Central Bank  (ECB). Here, Product and Money were represented by 3OC12HSL and C4HSL, the signaling molecules of the quorum sensing, respectively.</p></td>
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                 <td colspan="2"><h1>2.1 Molecular  Basis of Interdependence</h1></td>
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                 <td colspan="2"><h1>2.1 Molecular  Basis of Mutualism</h1></td>
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                 <td colspan="2"><p align="justify" class="info-18">We  designed two genetically engineered &lt;i&gt;E. coli&lt;/i&gt;, Company and  Customer (Fig. 2-1-3), each of which produces its own quorum sensing molecule  for the mutualism between the two. Since Company and Customer need  each other to continue the now-in-state economy, we designed the mutualism  between the two &lt;i&gt;E. coli&lt;/i&gt;. Company is dependent on the Money  supplied by Customer. The signaling molecule C4HSL represents Money. On the  other hand, Customer is dependent on the Product supplied by Company. The signaling  molecule 3OC12HSL represents the Product. The detailed design of the circuit is  shown in the following sections.</p></td>
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                 <td colspan="2"><p align="justify" class="info-18">We  designed two genetically engineered <em>E. coli</i></em>, Company and  Customer (Fig. 2-1-3), each of which produces its own quorum sensing molecule  for the mutualism between the two. Since Company and Customer need  each other to continue the now-in-state economy, we designed the mutualism  between the two <em>E. coli</em>. Company is dependent on the Money  supplied by Customer. On the  other hand, Customer is dependent on the Product supplied by Company. The detailed design of the circuit is  shown in the following sections.</br><a href="https://2014.igem.org/Team:Tokyo_Tech/Project/Animation1">Click here to see all picture of Animation</a></p></td>
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                 <td colspan="2"><p align="center" class="info-18"><a href="https://2014.igem.org/File:Tokyo_Tech_2-1-2.jpg"><img src="https://static.igem.org/mediawiki/2014/d/d8/Tokyo_Tech_Fig.2-1-2_Company_and_Customer_circuit_design.jpg" alt="" width="400" /></a></p></td>
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                 <td colspan="2"><p align="center" class="info-18"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig.2-1-2_Company_and_Customer_circuit_design.gif"><img src="https://static.igem.org/mediawiki/2014/a/a6/Tokyo_Tech_Fig.2-1-2_Company_and_Customer_circuit_design.gif" alt="" width="400" /></a></p></td>
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                 <td colspan="2"><div align="center">Fig. 2-1-3. The genetic circuit design of Company and Customer</div></td>
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                 <td colspan="2"><div align="center"><strong>Fig. 2-1-3.</strong> The genetic circuit design of Company and Customer</div></td>
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                 <td colspan="2">&nbsp;<a name="2.1.1" id="2.1.1"></a></td>
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                 <td colspan="2" class="head">2.1.1  Company</td>
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                 <td colspan="2" class="head">2.1.1. Company</td>
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                 <td><div align="center"><a href="https://2014.igem.org/wiki/index.php?title=Team:Tokyo_Tech/Project&action=edit"><img src="https://static.igem.org/mediawiki/2014/8/8c/Tokyo_Tech_The_genetic_circuit_Company.png" alt="" width="400" /></a></div></td>
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                 <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_The_genetic_circuit_Company.png"><img src="https://static.igem.org/mediawiki/2014/8/8c/Tokyo_Tech_The_genetic_circuit_Company.png" alt="" width="400" /></a></div></td>
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                 <td><div align="center">Fig. 2-1-4. The genetic  circuit design of Company</div></td>
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                 <td><div align="center"><strong>Fig. 2-1-4.</strong> The genetic  circuit design of Company</div></td>
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                 <td><div align="center">Fig. 2-1-5. The genetic  circuit design of Customer</div></td>
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                 <td><div align="center"><strong>Fig. 2-1-5.</strong> The genetic  circuit design of Customer</div></td>
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                 <td colspan="2"><h1 style="line-height:normal;">2.2. Native Prhl Promoter does not satisfy  <br />
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                 <td colspan="2"><h1 style="line-height:normal;">2.2 Native Prhl Promoter does not satisfy  <br />
                   requirement from system analysis</h1></td>
                   requirement from system analysis</h1></td>
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                 <td colspan="2"><p class="info-18">From the simulation results (Fig. 2-1-6), we  noticed that the strengths of Prhl and Plux promoter need to be equally strong  in order to promote the growth of the two &lt;i&gt;E. coli&lt;/i&gt;. If the  strength levels of Prhl and Plux promoters are in the red area, Company and  Customer can help the growth of each other. This means, both promoters need to  be strong and balanced for realizing the mutualism. However, if the strength  level is in the blue area, at least one of them cannot grow well. (See the <a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Overview">Modeling page</a> for detailed analysis).</p>                  </td>
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                 <td colspan="2"><p class="info-18">From the simulation results (Fig. 2-1-6), we  noticed that the strengths of Prhl and Plux promoter need to be equally strong  in order to promote the growth of the two <em>E. coli</em>. If the  strength levels of Prhl and Plux promoters are in the red area, Company and  Customer can help the growth of each other. This means, both promoters need to  be strong and balanced for realizing the mutualism. However, if the strength  level is in the blue area, at least one of them cannot grow well. (See the <a href="https://2014.igem.org/Team:Tokyo_Tech/Modeling/Overview">Modeling page</a> for detailed analysis).</p>                  </td>
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                 <td colspan="2"><div align="center">Fig. 2-1-6. The growth dependency of Prhl and Plux  promoters’ strength levels</div></td>
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                 <td colspan="2"><div align="center"><strong>Fig. 2-1-6.</strong> The growth dependency of Prhl and Plux  promoters’ strength levels</div></td>
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                 <td colspan="2"><p align="justify" class="info-18">  We then checked whether the strength levels of Prhl and Plux promoters satisfy  the conditions described above. As shown in Fig. 2-1-7, the strength level of  Plux promoter was about 20- fold higher than that of Plux promoter. Although  RBS strength modulation under Plux promoter might compensate the imbalance  between HSL-synsase expressions under the two promoters, such modulation  corresponds to the decreased Plux activity which did not lead to any growth of  Customer and Company. Therefore, the improvement of Prhl promoter’s strength  level became necessary to meet the modeling results.</p>
+
                 <td colspan="2"><p align="justify" class="info-18">  We then checked whether the strength levels of Prhl and Plux promoters satisfy  the conditions described above. As shown in Fig. 2-1-7, the strength level of  Plux promoter was about 20- fold higher than that of Plux promoter. Although  RBS strength modulation under Plux promoter might compensate the imbalance  between AHL-synthase expressions under the two promoters, such modulation  corresponds to the decreased Plux activity which did not lead to any growth of  Customer and Company. Therefore, the improvement of Prhl promoter’s strength  level became necessary to meet the modeling results.</p>
                   <div>
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-
                 <td colspan="2"><div align="center"> 2-1-7. The result of Prhl, Plux promoter assay</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-7.</strong> The result of Prhl, Plux promoter assay</div></td>
               </tr>
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               </tr>
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-
                 <td colspan="2"><span class="info-18">To meet the modeling results, we added three improved C4HSL-dependent promoters with high maximum expression  level by combinations of regulatory-protein binding sites (Fig. 2-1-8).</span></td>
+
                 <td colspan="2"><span class="info-18">To meet the modeling results, we added three improved C4HSL-dependent promoters with high maximum expression  level by combinations of regulatory-protein binding sites. (Fig. 2-1-8)</span></td>
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                 <td colspan="2"><div align="center">Fig. 2-1-8. Designs of  improved Prhl promoters</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-8.</strong> Designs of  improved Prhl promoters</div></td>
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                 <td colspan="2"><p align="justify" class="info-18">First, we designed a new Lux promoter with two RhlR  binding sites instead of two LuxR binding sites (Prhl(RR): <a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>) as tried in a previous paper (Chuang 2009). To  evaluate the function of this promoter, we constructed Prhl(RR)-GFP plasmids  and measured the fluorescence intensity by flow cytometer. In the measurement,  we confirmed that GFP under the control of Prhl(RR) promoter showed about  20-fold higher in the fluorescence than that of the native Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) (See  the <a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay">Experiment page</a>)  (Fig. 2-1-9).</p></td>
+
                 <td colspan="2"><p align="justify" class="info-18">First, we designed a new Lux promoter with two RhlR  binding sites instead of two LuxR binding sites (Prhl(RR): <a href="http://parts.igem.org/Part:BBa_K1529320">BBa_K1529320</a>) as tried in a previous paper (Chuang 2009). To  evaluate the function of this promoter, we constructed Prhl(RR)-GFP plasmids  and measured the fluorescence intensity by flow cytometer. In the measurement,  we confirmed that GFP under the control of Prhl(RR) promoter showed about  20-fold higher in the fluorescence than that of the native Prhl promoter (<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>) (See  the <a href="https://2014.igem.org/Team:Tokyo_Tech/Experiment/Prhl_reporter_assay">Experiment page</a>)  (Fig. 2-1-9)</p></td>
                 </tr>
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                 <td colspan="2"><p class="info-18">One of our new promoter, Prhl(RL) (Fig. 2-1-9 lane  4), improved in its expression level while keeping the low leakage. GFP under  the control of this Prhl(RL) promoter showed about 82-fold higher in the  fluorescence with C4 addition compared to the fluorescence without C4 addition.  This is much higher than that of the native Prhl promoter, which is 22-fold.</p></td>
+
                 <td colspan="2"><p class="info-18">One of our new promoter, Prhl(RL) (Fig. 2-1-9.lane  4), improved in its expression level while keeping the low leakage. GFP under  the control of this Prhl(RL) promoter showed about 82-fold higher in the  fluorescence with C4 addition compared to the fluorescence without C4 addition.  This is much higher than that of the native Prhl promoter, which is 22-fold.</p></td>
               </tr>
               </tr>
               <tr>
               <tr>
-
                 <td colspan="2"><p class="info-18">Although the other Prhl(LR) promoter (Fig. 2-1-9 lane 3) showed a higher maximum expression level, it showed a significant leak  like Prhl(RR) promoter. GFP under the control of Prhl(LR) promoter showed about  17-fold higher in the fluorescence with C4 addition compared to the  fluorescence without C4 addition. However, the leak showed no less than 25-fold  higher than the native Prhl promoter. Thus we used our most improved Prhl(RL) (K1529320)  in the following experiments and modeling.</p></td>
+
                 <td colspan="2"><p class="info-18">Although the other Prhl(LR) promoter (Fig. 2-1-9. lane 3) showed a higher maximum expression level, it showed a significant leak  like Prhl(RR) promoter(Fig. 2-1-9. lane  2). GFP under the control of Prhl(LR) promoter showed about  17-fold higher in the fluorescence with C4 addition compared to the  fluorescence without C4 addition. However, the leak showed no less than 25-fold  higher than the native Prhl promoter. Thus we used our most improved Prhl(RL) (K1529320)  in the following experiments and modeling.</p></td>
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                 <td colspan="2"><div align="center">
                 <td colspan="2"><div align="center">
-
                   <div align="center">Fig. 2-1-9. The improvement of  Prhl promoters</div>
+
                   <div align="center"><strong>Fig. 2-1-9.</strong> The improvement of  Prhl promoters</div>
                 </div></td>
                 </div></td>
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                 <td colspan="2"><h1 style="line-height:normal;">2.4. HSL-dependent  responses of Company <em><i>E. coli</i></em> with improved promoter and Customer <em><i>E. coli</i></em></h1>
+
                 <td colspan="2"><h1 style="line-height:normal;">2.4. AHL-dependent  responses of Company <em>E. coli</em> with improved promoter and Customer <em>E. coli</em></h1>
                 <a name="2.4.1" id="2.4.1"></a></td>
                 <a name="2.4.1" id="2.4.1"></a></td>
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               <tr>
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-
                 <td colspan="2" class="head">2.4.1.  Company</td>
+
                 <td colspan="2" class="head">2.4.1.  Company with improved promoter</td>
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-
                 <td colspan="2"><div align="center">Fig.2-1-10 Gene circuit of Company with  
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-10.</strong> Genetic circuit of Company with  
                 improved promoter</div></td>
                 improved promoter</div></td>
               </tr>
               </tr>
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                 </tr>
                 </tr>
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-
                 <td colspan="2"><p class="info-18">For  construction of the C4HSL-dependent chloramphenicol resistance gene product  (CmR) and 3OC12HSL production module, we designed a new part Prhl(RL)-CmR-LasI.  (BBa_K1529302) (Fig. 2-1-10). In order to confirm the Company’s dependency on  C4HSL, we measured the growth of Company cell in the presence and absence of  C4HSL. After the induction, we added chloramphenicol into the medium and  measured the optical density for about 10 hours to estimate the concentration of the cell.</p></td>
+
                 <td colspan="2"><p class="info-18">For  construction of the C4HSL-dependent chloramphenicol resistance gene product  (CmR) and 3OC12HSL production module, we designed a new part Prhl(RL)-CmR-LasI.  (BBa_K1529302) (Fig. 2-1-10) In order to confirm the Company’s dependency on  C4HSL, we measured the growth of Company cell in the presence and absence of  C4HSL. After the induction, we added chloramphenicol into the medium and  measured the optical density for about 10 hours to estimate the concentration of the cell.</p></td>
                 </tr>
                 </tr>
                
                
               <tr>
               <tr>
-
                 <td colspan="2"><p class="info-18">Without  induction of C4HSL, the cell cannot express CmR resistance gene and cannot  survive in the presence of chloramphenicol. As shown in Fig. 2-1-11, when C4HSL  is added to the culture, Company cell survived and increased. This result indicates that CmR was produced in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI.</p>                  <a href="https://2014.igem.org/File:Tokyo_Tech_2-1-11.jpg"></a></td>
+
                 <td colspan="2"><p class="info-18">Without  induction of C4HSL, the cell cannot express CmR resistance gene and cannot  survive in the presence of chloramphenicol. As shown in Fig. 2-1-11, when C4HSL  is added to the culture, Company cell survived and increased. This result indicates that Company cell produced 3OC12HSL in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI. From these
 +
experiments, we confirmed that a new part Prhl(RL)-CmR-LasI synthesized CmR and 3OC12HSL work as expected.</p>                   </td>
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-
                <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Company_can_not_grow_1.png"><img src="https://static.igem.org/mediawiki/2014/e/e4/Tokyo_Tech_Company_can_not_grow_1.png" alt="" width="445" /></a></div></td>
+
                                  <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Company_can_not_grow_2.png"><img src="https://static.igem.org/mediawiki/2014/7/78/Tokyo_Tech_Company_can_not_grow_2.png" alt="" width="700" /></a></div></td>
-
                <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Company_can_not_grow_2.png"><img src="https://static.igem.org/mediawiki/2014/7/78/Tokyo_Tech_Company_can_not_grow_2.png" alt="" width="445" /></a></div></td>
+
               </tr>
               </tr>
               <tr>
               <tr>
-
                 <td colspan="2"><div align="center">Fig.2-1-11. Company  cannot grow without C4HSL</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-11.</strong> Company  cannot grow without C4HSL</div></td>
                 </tr>
                 </tr>
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-
                 <td colspan="2"><p class="info-18">To characterize the function of C4HSL-dependent  3OC12HSL production, we also performed a reporter assay by using lux reporter cell  (Fig. 2-1-12). First, the expression of LasI was induced by adding C4HSL to the  culture of the Company cell. Then, the supernatant of the culture was added to  the culture of reporter cell. The expression  of GFP in the reporter cell was measured by flow cytometer.</p></td>
+
                 <td colspan="2"><p class="info-18">To characterize the function of C4HSL-dependent  3OC12HSL production, we also performed a reporter assay by using lux reporter cell  (Fig. 2-1-12) First, the expression of LasI was induced by adding C4HSL to the  culture of the Company cell. Then, the supernatant of the culture was added to  the culture of reporter cell. The expression  of GFP in the reporter cell was measured by flow cytometer.</p></td>
                 </tr>
                 </tr>
               </table>
               </table>
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               </tr>
               </tr>
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                 <td><p align="justify" class="info-18">As  Fig. 2-1-11 shows, when the reporter cell E was incubated in the culture of the  induced Company cell, the fluorescence intensity of the reporter cell  increased. Comparing the results of the culture with the induced Company cell  and not induced Company cell, the reporter cell in the supernatant of induced  cell had 29-fold higher fluorescence intensity.</p></td>
+
                 <td><p align="justify" class="info-18">As  Fig. 2-1-12 shows, when the reporter cell E was incubated in the culture of the  induced Company cell, the fluorescence intensity of the reporter cell  increased. Comparing the results of the culture with the induced Company cell  and not induced Company cell, the reporter cell in the supernatant of induced  cell had 29-fold higher fluorescence intensity.</p></td>
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                 <td><div align="center">Fig.2-1-12 Company  excretes 3OC12HSL by new BioBrick parts when C4HSL exists</div></td>
+
                 <td><div align="center"><strong>Fig. 2-1-12.</strong> Company  excretes 3OC12HSL by new BioBrick parts when C4HSL exists</div></td>
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-
                 <td colspan="2"><p align="justify" class="info-18">For  construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL  production module, we designed a new part Plux-CmR-RhlI (BBa_K1529797). In  order to confirm the Customer’s dependency on 3OC12HSL, we measured the growth  of Customer cell in the presence and absence of 3OC12HSL. After induction, we  added chloramphenicol into the medium and measured optical density for about ten  hours to estimate the concentration of the  cell.</p></td>
+
                 <td colspan="2"><p align="justify" class="info-18">For  construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL  production module, we designed a new part Plux-CmR-RhlI (BBa_K1529797)(Fig. 2-1-13). In  order to confirm the Customer’s dependency on 3OC12HSL, we measured the growth  of Customer cell in the presence and absence of 3OC12HSL. After induction, we  added chloramphenicol into the medium and measured optical density for about ten  hours to estimate the concentration of the  cell.</p></td>
               </tr>
               </tr>
               <tr>
               <tr>
-
                 <td colspan="2"><p align="justify" class="info-18">Without  induction of 3OC12HSL, the cell cannot express CmR and cannot survive in the  presence of chloramphenicol. As shown in Fig. 2-1-13, when 3OC12HSL was added  to the culture, Customer cell survived and increased. This result indicates  that CmR was produced in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.</p></td>
+
                 <td colspan="2"><p align="justify" class="info-18">Without  induction of 3OC12HSL, the cell cannot express CmR and cannot survive in the  presence of chloramphenicol. As shown in Fig. 2-1-14, when 3OC12HSL was added  to the culture, Customer cell survived and increased. This result indicates  that CmR was produced in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.</p></td>
               </tr>
               </tr>
-
             
 
               <tr>
               <tr>
                 <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Genetic_circuit_design_of_customer.png"><img src="https://static.igem.org/mediawiki/2014/thumb/6/6c/Tokyo_Tech_Genetic_circuit_design_of_customer.png/800px-Tokyo_Tech_Genetic_circuit_design_of_customer.png" width="500" /></a></div>                   
                 <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Genetic_circuit_design_of_customer.png"><img src="https://static.igem.org/mediawiki/2014/thumb/6/6c/Tokyo_Tech_Genetic_circuit_design_of_customer.png/800px-Tokyo_Tech_Genetic_circuit_design_of_customer.png" width="500" /></a></div>                   
-
                 <div align="center">Fig. 2-1-13. Genetic  circuit design of Customer</div></td>
+
                 <div align="center"><strong>Fig. 2-1-13.</strong> Genetic  circuit design of Customer</div></td>
                 </tr>
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                 <td width="445"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Customer_cannot_survive_1.png"><img src="https://static.igem.org/mediawiki/2014/d/df/Tokyo_Tech_Customer_cannot_survive_1.png" width="400" /></a></div></td>
+
                 <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Customer_cannot_survive_1.png"><img src="https://static.igem.org/mediawiki/2014/d/df/Tokyo_Tech_Customer_cannot_survive_1.png" width="700" /></a></div></td>
-
                <td width="445"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Customer_cannot_survive_2.png"><img src="https://static.igem.org/mediawiki/2014/5/55/Tokyo_Tech_Customer_cannot_survive_2.png" width="400" /></a></div></td>
+
             
               </tr>
               </tr>
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               <tr>
-
                 <td colspan="2"><div align="center">Fig. 2-1-14. Customer  cannot survive without 3OC12HSL</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-14.</strong> Customer  cannot survive without 3OC12HSL</div></td>
                 </tr>
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               </tr>
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-
                 <td colspan="2"><p class="info-18">To characterize the function of 3OC12HSL-dependent  C4HSL production, we also performed a reporter assay by using lux reporter cell  (Fig. 2-1-14).</p></td>
+
                 <td colspan="2"><p class="info-18">To characterize the function of 3OC12HSL-dependent  C4HSL production, we also performed a reporter assay by using lux reporter cell  (Fig. 2-1-15).</p></td>
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                 <td colspan="2"><p align="justify" class="info-18">As  Fig. 2-1-14 shows, when the reporter cell Plux-CmR-RhlI was incubated in the  culture of the induced Customer cell, the fluorescence intensity of the  reporter cell increased. Comparing the results of the induced cell and not  induced dell, reporter cell in the supernatant of the induced cell had 95-fold  higher fluorescence intensity. This result indicates that Customer cell  produced C4HSL in response to 3OC12HSL induction by the function of  Plux-CmR-RhlI.</p></td>
+
                 <td colspan="2"><p align="justify" class="info-18">As  Fig. 2-1-15 shows, when the reporter cell Plux-CmR-RhlI was incubated in the  culture of the induced Customer cell, the fluorescence intensity of the  reporter cell increased. Comparing the results of the induced cell and not  induced dell, reporter cell in the supernatant of the induced cell had 95-fold  higher fluorescence intensity. This result indicates that Customer cell  produced C4HSL in response to 3OC12HSL induction by the function of  Plux-CmR-RhlI.</p></td>
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                 <td colspan="2"><div align="center">Fig.2-1-15. Customer  excretes C4HSL when C12HSL exists</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-15.</strong> Customer  excretes C4HSL when C12HSL exists</div></td>
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                 <td><div align="center">Fig. 2-1-16. The result of co-culture assay</div></td>
+
                 <td><div align="center"><strong>Fig. 2-1-16.</strong> The result of co-culture assay</div></td>
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                 <td colspan="2"><span class="info-18">     
                 <td colspan="2"><span class="info-18">     
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                 </span>                  <p class="info-18">The result of the co-culture assay is shown  in Fig. 2-1-15. By looking at the Company cells expressing GFP, the OD increased with the co-cultured cells than Company cells. From this point, we  can say that Company and Customer actually mutualize in the medium.</p></td>
+
                 </span>                  <p class="info-18">The result of the co-culture assay is shown  in Fig. 2-1-16. By looking at the fluorescence intensity of GFP in Company cells, the optical density increased faster in co-culture than single culture. From this point, we  can say that Company and Customer actually mutualize in the medium.</p></td>
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                 <td colspan="2"><p align="justify" class="info-18">    We  noticed that the introduction of bank may assist not only the symbiosis with small  amount of cells in a test tube, but also the understanding of economics as an education  tool. In real economy, a central bank will supply money to the market when money  is in short supply. Therefore, we designed Bank <em><i>E. coli</i></em> that  does the same work as central bank.</p></td>
+
                 <td colspan="2"><p align="justify" class="info-18">    We  noticed that the introduction of bank may assist not only the symbiosis with small  amount of cells in a test tube, but also the understanding of economics as an education  tool. In real economy, a central bank will supply money to the market when money  is in short supply. Therefore, we designed Bank <em>E. coli</em> that  does the same work as central bank.</p></td>
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                 <td colspan="2"><h1>3.2. Genetic  circuit design of Bank <i>E. coli</i></h1></td>
+
                 <td colspan="2"><h1>3.2. Genetic  circuit design of Bank <em>E. coli</em></h1></td>
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                 <td colspan="2"><div align="center">Fig. 2-1-17. Genetic  circuit design of Bank <i>E. coli</i></div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-17.</strong> Genetic  circuit of Bank <em>E. coli</em></div></td>
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                 <td colspan="2"><p align="justify" class="info-18">The circuit  design of Bank <em><i>E. coli</i></em> is shown in Fig. 2-1-16. Bank <em><i>E. coli</i></em> functions  as a central bank to regulate the money supply in the market. Bank <em><i>E. coli</i></em> can change into two states by using  toggle switch (Gardner, 2000), which depends on C4HSL concentration (Fig.  2-1-17).</p></td>
+
                 <td colspan="2"><p align="justify" class="info-18">The circuit  design of Bank <em>E. coli</em> is shown in Fig. 2-1-16 Bank <em>E. coli</em> functions  as a central bank to regulate the money supply in the market. Bank <em>E. coli</em> can change into two states by using  toggle switch (Gardner, 2000), which depends on C4HSL concentration (Fig.  2-1-17).</p></td>
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                 <td><div align="center">Fig.2-1-18 Distribution  state</div></td>
+
                 <td><div align="center"><strong>Fig. 2-1-18.</strong> Distribution  state</div></td>
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                 <td height="19"><div align="center">Fig.2-1-19 Change to  collection state</div></td>
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                 <td height="19"><div align="center"><strong>Fig. 2-1-19.</strong> Change to  collection state</div></td>
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                 <td height="19" rowspan="2"><p class="info-18">If the  amount of money in the economy become excessive, Bank will be in the collection  state. In this state, AiiA in Bank will be expressed. AiiA decomposes C4HSL and  3OC12HSL in the medium to decrease the money supply in the economy.</p></td>
+
                 <td height="19" rowspan="2"><p class="info-18">If the  amount of money in the economy becomes excessive, Bank will be in the collection  state. In this state, AiiA in Bank will be expressed. AiiA decomposes C4HSL and  3OC12HSL in the medium to decrease the money supply in the economy.</p></td>
                 <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_2-1-25.jpg"><img src="https://static.igem.org/mediawiki/2014/8/80/Tokyo_Tech_2-1-25.jpg" alt="" width="300" /></a></div></td>
                 <td><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_2-1-25.jpg"><img src="https://static.igem.org/mediawiki/2014/8/80/Tokyo_Tech_2-1-25.jpg" alt="" width="300" /></a></div></td>
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                 <td><div align="center">Fig.2-1-20 Collection  State</div></td>
+
                 <td><div align="center"><strong>Fig. 2-1-20.</strong> Collection  State</div></td>
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                 <td><div align="center">Fig.2-1-21. Change to  distribution state</div></td>
+
                 <td><div align="center"><strong>Fig. 2-1-21.</strong> Change to  distribution state</div></td>
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                 <td colspan="2"><h1>3.3. Modeling; Bank actually helps the development  of the economy</h1></td>
+
                 <td colspan="2"><h1>3.3. Modeling: Bank actually helps the development  of the economy</h1></td>
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                 <td colspan="2"><p class="info-18"> We made a  mathematical model to ensure the functioning of the Bank. Bank helps Company  and Customer to grow well even with a few amounts. As shown in Fig. 2-1-26, if  the amounts of Company and Customer are few, then they cannot grow well. But  once the Bank is included, all of them can grow well.</p></td>
+
                 <td colspan="2"><p class="info-18"> We made a  mathematical model to ensure the functioning of the Bank. Bank helps Company  and Customer to grow well even with a few amounts. As shown in Fig. 2-1-22, if  the amounts of Company and Customer are few, then they cannot grow well. But  once the Bank is included, all of them can grow well.</p></td>
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                 <td colspan="2"><div align="center">Fig. 2-1-22. Bank helps Company and Customer to  grow well</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-22.</strong> Bank helps Company and Customer to  grow well</div></td>
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                 <td colspan="2"><p class="info-18">Also  to ensure the functioning, we analyzed the Bank’s internal switch depending on  the C4HSL concentration. Fig. 2-1-17 shows the RhlI concentration depending on  the C4 concentration in the Bank cell. When the C4 concentration increases from  low to high, the RhlI concentration follows the green line on the figure,  decreasing the RhlI expression. This means the Bank switches its state from  distribution state to collection state. On the other hand, when the C4  concentration decreases, the RhlI concentration follows the blue line,  increasing the RhlI expression. This means the Bank switches its state from  collection state to distribution state.</p></td>
+
                 <td colspan="2"><p class="info-18">Also  to ensure the functioning, we analyzed the Bank’s internal switch depending on  the C4HSL concentration. Fig. 2-1-23 shows the RhlI concentration depending on  the C4 concentration in the Bank cell. When the C4 concentration increases from  low to high, the RhlI concentration follows the green line on the figure,  decreasing the RhlI expression. This means the Bank switches its state from  distribution state to collection state. On the other hand, when the C4  concentration decreases, the RhlI concentration follows the blue line,  increasing the RhlI expression. This means the Bank switches its state from  collection state to distribution state.</p></td>
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                 <td colspan="2"><div align="center">Fig. 2-1-23. Bank changes its state from  Distribution State to Collection State</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-23.</strong> Bank changes its state from  Distribution State to Collection State</div></td>
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                 <td colspan="2"><h2 style="line-height:normal;">4. Policy and Practice  suggested introduction of Economic Wave</h2></td>
+
                 <td colspan="2"><h2 style="line-height:normal;">4. Economic Wave: Advice from Entrepreneurs</h2></td>
               </tr>
               </tr>
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                 <td colspan="2"><p class="info-18">We introduced our Bank <em>E. coli</em> project to  businessmen, executives, and technicians engaged in IT companies at a workshop  called MUSE TALK for professional opinions (See <a href="https://2014.igem.org/Team:Tokyo_Tech/Policy_and_Practices#MUSE">Policy  and Practice</a> for more details). They  pointed out that economic wave should be integrated into our system to make it  more realistic (Fig. 2-1-28).</p></td>
+
                 <td colspan="2"><p class="info-18">We introduced our Bank <em>E. coli</em> project to  businessmen, executives, and technicians engaged in IT companies at a workshop  called MUSE TALK for professional opinions (See <a href="https://2014.igem.org/Team:Tokyo_Tech/Policy_and_Practices#MUSE">Policy  and Practice</a> for more details). They  pointed out that economic wave should be integrated into our system to make it  more realistic (Fig. 2-1-24).</p></td>
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                 <td colspan="2"><div align="center">Fig. 2-1-24.  Our teammates exchanging opinions with professionals</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-24.</strong> Our teammates exchanging opinions with professionals</div></td>
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                 <td colspan="2"><p class="info-18"> In order to develop  our educational tool for iGMERs more, we merged the idea of economic wave into  our system as the fluctuation of C4HSL concentration. Even though Company and  Customer can grow well only by themselves as shown in Fig. 2-1-29(left), they  cannot endure the effect of economic wave as shown in Fig. 2-1-29(center).  These results show that Company and Customer are not good at dealing with the economic  wave. Let us introduce the Bank to the market with  economic wave. As shown in Fig. 2-1-29(right), Bank helps Company and Customer  to grow well. </p></td>
+
                 <td colspan="2"><p class="info-18"> In order to develop  our educational tool for iGEMERs more, we merged the idea of economic wave into  our system as the fluctuation of C4HSL concentration. Even though Company and  Customer can grow well only by themselves as shown in Fig. 2-1-25(left), they  cannot endure the effect of economic wave as shown in Fig. 2-1-25(center).  These results show that Company and Customer are not good at dealing with the economic  wave. Let us introduce the Bank to the market with  economic wave. As shown in Fig. 2-1-25(right), Bank helps Company and Customer  to grow well. </p></td>
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                   <td colspan="3"><div align="center">Fig. 2-1-25 Company and Customer cannot survive  when they face economic wave. </div></td>
+
                   <td colspan="3"><div align="center"><strong>Fig. 2-1-25.</strong> Company and Customer cannot survive  when they face economic wave. </div></td>
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                 <td colspan="2"><p class="info-18">Money supply in these situations, represented by C4  concentrations, are shown in Fig. 2-1-30. Even though the money supply  decreases very much by the effect of economic wave as shown in  Fig,2-1-30(center), the introduction of the Bank changes the state drastically,  results in abundant money supply(Fig. 2-1-30(right)).</p></td>
+
                 <td colspan="2"><p class="info-18">Money supplies in these situations, represented by C4  concentrations, are shown in Fig. 2-1-26. Even though the money supply  decreases very much by the effect of economic wave as shown in  Fig,2-1-26(center), the introduction of the Bank changes the state drastically,  results in abundant money supply Fig. 2-1-26(right).</p></td>
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                   <td colspan="3"><div align="center">Fig. 2-1-26 Money supply (C4 concentration) in every  situation described in Fig. 2-1-29.</div></td>
+
                   <td colspan="3"><div align="center"><strong>Fig. 2-1-26</strong> Money supply (C4 concentration) in every  situation described in Fig. 2-1-25.</div></td>
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                 <td colspan="2"><p class="info-18">Although the above results show how Bank stabilize  the system, small change of parameter in economic wave makes Bank to fail  managing.  With harsh economic wave, the effect of economic wave begins to be out  of hands of Bank. As shown in Fig.2-1-32(left), the population of Company and  Customer fluctuates very much. This is due to the fluctuation of money supply in  the market(Fig. 2-1-32(right)). Interestingly, the Bank constantly grows while  the other two suffers the fluctuation of the money supply (Fig. 2-1-32(left)). Be  carful not to be like this in your countries.</p></td>
+
                 <td colspan="2"><p class="info-18">Although the above results show how Bank stabilizes the system, small change of parameter in economic wave makes Bank to fail  managing.  With harsh economic wave, the effect of economic wave begins to be out  of hands of Bank. As shown in Fig.2-1-27(left), the population of Company and  Customer fluctuates very much. This is due to the fluctuation of money supply in  the market(Fig. 2-1-27(right)). Interestingly, the Bank constantly grows while  the other two suffers the fluctuation of the money supply (Fig. 2-1-27(left)). Be  careful not to be like this in your countries.</p></td>
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                 <td colspan="2"><div align="center">Fig. 2-1-27 Bank can help Company and Customer even  with harsh economic wave?</div></td>
+
                 <td colspan="2"><div align="center"><strong>Fig. 2-1-27.</strong> Bank can help Company and Customer even  with harsh economic wave</div></td>
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                 <td colspan="2" class="entry-long">&nbsp;</td>
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                 <td colspan="2" class="entry-long">&nbsp;<a name="5" id="5"></a></td>
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                 <td colspan="2">&nbsp;<a name="5" id="5"></a></td>
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                 <td colspan="2">&nbsp;</td>
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                <td colspan="2"><h2>5. Reference </h2></td>
+
                        <td colspan="2"><h2>5. Project Evaluation</h2></td>
 +
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                        <td colspan="2">&nbsp;</td>
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                        <td colspan="2"><p align="left" class="info-18">Finally, the evaluation of our educational tool using E. coli and
 +
BioBrick showed that our project is highly effective for students majoring in biology to understand economics (Fig. 2-1-28).</p></td>
 +
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                        <td colspan="2">&nbsp;</td>
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                        <td colspan="2"><div align="center"><a href="https://2014.igem.org/File:Tokyo_Tech_Fig1-1-9.png"><img src="https://static.igem.org/mediawiki/2014/a/a0/Tokyo_Tech_Fig1-1-9.png" width="615" height="243" /></a></div></td>
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                        <td colspan="2"><div align="center"><strong>Fig. 2-1-28. </strong>Effects of our presentation</div></td>
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                        <td colspan="2">&nbsp;</td>
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                        <td colspan="2"><div align="right" class="info-18">(<a href="https://2014.igem.org/Team:Tokyo_Tech/Policy_and_Practices">Go to Policy and Practices Page</a>)</div></td>
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                        <td colspan="2" class="entry-long">&nbsp;<a name="6" id="6"></a></td>
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                <td colspan="2">&nbsp;</td>
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                <td colspan="2"><h2>6. Reference </h2></td>
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                 <td colspan="2" class="info-18">10. Kendall M. Gray <em>et al.</em> (1994) Interchangeability and  specificity of components from the quorum-sensing regulatory systems of <em>Vibrio fischeri</em> and <em>Pseudomonas aeruginosa</em>. Journal of Bacteriology 176(10): 3076–3080</td>
                 <td colspan="2" class="info-18">10. Kendall M. Gray <em>et al.</em> (1994) Interchangeability and  specificity of components from the quorum-sensing regulatory systems of <em>Vibrio fischeri</em> and <em>Pseudomonas aeruginosa</em>. Journal of Bacteriology 176(10): 3076–3080</td>
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                <td colspan="2" class="info-18">11. Natthawut  Wiriyathanawudhiwong <em>et al.</em> (2009)  The outer membrane TolC is involved in cysteine tolerance and overproduction in <em>Escherichia coli. </em>Appl Microbiol  Biotechnol 81: 903-913</td>
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                <td colspan="2" class="info-18">12. McIver CJ <em>et al.</em> (1987) Cysteine requirements of  naturally occurring cysteine auxotrophs of <em>Escherichia  coli</em>. Pathology 19(4): 361-363</td>
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Latest revision as of 04:00, 18 October 2014

Tokyo_Tech

Project

Learning economics by Bank E. coli

Contents

1. Project Planning: Interaction with General Public

2. Mutualism between Company and Customer

2.1 Molecular Basis of Mutualism

2.1.1 Company

2.1.2 Customer

2.2 Native Prhl promoter does not satisfy requirement from system analysis

2.3 The improvement of Prhl promoter

2.4 AHL-dependent responses of Company E. coli with improved promoter and Customer E. coli

2.4.1 Company with improved promoter

2.4.2 Customer

2.5 Assay of symbiosis between Company and Customer by co-culture

3. Addition of Bank

3.1 Motivation

3.2 Genetic circuit design of Bank E. coli

3.2.1 Distribution state

3.2.2 Change to Collection State

3.2.3 Collection State

3.2.4 Change to distribution state

3.3 Modeling:Bank actually helps the development of economy

4. Economic Wave: Advice from Entrepreneurs

5. Project Evaluation

6. Reference

 
 
 

1.Project Planning: Interaction with General Public

Fig. 2-1-1. Our members discussing about the project with the visitor
 

This year, we decided to make an educational tool for economics by using E. coli. This tool, which is supported with BioBrick parts and modeling, can be easily adapted to not only iGEMers, but also other students majoring in biology who are great human resource of innovation.

At the project planning stage of this year, we took part in a poster session held at the school festival of the University of Tokyo. We showed our preliminary plans, such as E. coli solar battery, and fertilizer. (See Policy and Practice for more details) (Fig. 2-1-1). After communicating with people engaged in business, we realized that we do not know much about economic system. This lack of knowledge, which may be common with other iGEMers, can be an obstacle for innovation from our research activity. In order to solve this, we thought of making an educational tool for economics by using E. coli and BioBrick, which are familiar for iGEMers.

 
Fig. 2-1-2. Relationship among three types of E. coli
 

Our educational tool has three types of E. coli. They are Bank, Company, and Customer (Fig. 2-1-2).Like the exchange of money and products in the real economy, we designed these three E. coli to exchange Product and Money in the tool’s economic system. The Company makes Product, and sells them to the Customer. On the other hand, the Customer pays Money to buy the Product made by the Company. With this mutual action, the Customer and the Company build mutualistic relationship. Additionally, the Bank regulates the Money supply in the market. It functions as a central bank like FRB and European Central Bank (ECB) (see here for more information about central bank). Here, Product and Money were represented by 3OC12HSL and C4HSL, the signaling molecules of the quorum sensing, respectively.

 
 
 

2. Mutualism between Company and Customer

 

2.1 Molecular Basis of Mutualism

We designed two genetically engineered E. coli, Company and Customer (Fig. 2-1-3), each of which produces its own quorum sensing molecule for the mutualism between the two. Since Company and Customer need each other to continue the now-in-state economy, we designed the mutualism between the two E. coli. Company is dependent on the Money supplied by Customer. On the other hand, Customer is dependent on the Product supplied by Company. The detailed design of the circuit is shown in the following sections.
Click here to see all picture of Animation

 

Fig. 2-1-3. The genetic circuit design of Company and Customer
 
2.1.1. Company
 

In the presence of C4HSL, which represents Money, Company can produce chloramphenicol-resistance gene product (CmR) and LasI. CmR protects the Company from the antibiotic action of chloramphenicol. LasI produces signaling molecule 3OC12HSL, which represents the Product made by the Company. If there is not any C4HSL in the medium, Company cannot produce CmR. This will lead to the growth inhibition of the Company, which represents the Company’s bankruptcy.

Fig. 2-1-4. The genetic circuit design of Company
 
   
2.1.2 Customer  

The basic design of Customer’s circuit is the same as Company. In the presence of 3OC12HSL, which represents the Product, Customer produces CmR and RhlI. CmR prevents the Customer from growth inhibition, and RhlI produces C4HSL, which represents Money. If there is not any Product in the market, Customer cannot produce CmR. This leads to the growth inhibition of Customer.

 
Fig. 2-1-5. The genetic circuit design of Customer
   

2.2 Native Prhl Promoter does not satisfy
requirement from system analysis

From the simulation results (Fig. 2-1-6), we noticed that the strengths of Prhl and Plux promoter need to be equally strong in order to promote the growth of the two E. coli. If the strength levels of Prhl and Plux promoters are in the red area, Company and Customer can help the growth of each other. This means, both promoters need to be strong and balanced for realizing the mutualism. However, if the strength level is in the blue area, at least one of them cannot grow well. (See the Modeling page for detailed analysis).

 
Fig. 2-1-6. The growth dependency of Prhl and Plux promoters’ strength levels
 

We then checked whether the strength levels of Prhl and Plux promoters satisfy the conditions described above. As shown in Fig. 2-1-7, the strength level of Plux promoter was about 20- fold higher than that of Plux promoter. Although RBS strength modulation under Plux promoter might compensate the imbalance between AHL-synthase expressions under the two promoters, such modulation corresponds to the decreased Plux activity which did not lead to any growth of Customer and Company. Therefore, the improvement of Prhl promoter’s strength level became necessary to meet the modeling results.

 
Fig. 2-1-7. The result of Prhl, Plux promoter assay
 

2.3. The improvement of Prhl promoter

To meet the modeling results, we added three improved C4HSL-dependent promoters with high maximum expression level by combinations of regulatory-protein binding sites. (Fig. 2-1-8)
 
Fig. 2-1-8. Designs of improved Prhl promoters
 

First, we designed a new Lux promoter with two RhlR binding sites instead of two LuxR binding sites (Prhl(RR): BBa_K1529320) as tried in a previous paper (Chuang 2009). To evaluate the function of this promoter, we constructed Prhl(RR)-GFP plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl(RR) promoter showed about 20-fold higher in the fluorescence than that of the native Prhl promoter (BBa_R0071) (See the Experiment page) (Fig. 2-1-9)

However, Prhl(RR) promoter showed a significant leak in the absence of C4HSL(See the Experiment page). High level of leakage is not suitable for the Company-Customer relationship because their mutualism will be broken.

In order to lessen the leak while keeping higher expression level than the native PRhl promoter, we newly designed two promoters, Prhl(LR) (BBa_K1529310) and Prhl(RL) (BBa_K1529300). These promoters have one LuxR binding site and one RhlR binding site. We changed either the former RhlR binding site of Prhl(RR) promoter to LuxR binding site (Prhl(LR)), or the latter RhlR binding site to Lux binding site (Prhl(RL)).

One of our new promoter, Prhl(RL) (Fig. 2-1-9.lane 4), improved in its expression level while keeping the low leakage. GFP under the control of this Prhl(RL) promoter showed about 82-fold higher in the fluorescence with C4 addition compared to the fluorescence without C4 addition. This is much higher than that of the native Prhl promoter, which is 22-fold.

Although the other Prhl(LR) promoter (Fig. 2-1-9. lane 3) showed a higher maximum expression level, it showed a significant leak like Prhl(RR) promoter(Fig. 2-1-9. lane 2). GFP under the control of Prhl(LR) promoter showed about 17-fold higher in the fluorescence with C4 addition compared to the fluorescence without C4 addition. However, the leak showed no less than 25-fold higher than the native Prhl promoter. Thus we used our most improved Prhl(RL) (K1529320) in the following experiments and modeling.

 
Fig. 2-1-9. The improvement of Prhl promoters
 

2.4. AHL-dependent responses of Company E. coli with improved promoter and Customer E. coli

2.4.1. Company with improved promoter
 
Fig. 2-1-10. Genetic circuit of Company with improved promoter
 

For construction of the C4HSL-dependent chloramphenicol resistance gene product (CmR) and 3OC12HSL production module, we designed a new part Prhl(RL)-CmR-LasI. (BBa_K1529302) (Fig. 2-1-10) In order to confirm the Company’s dependency on C4HSL, we measured the growth of Company cell in the presence and absence of C4HSL. After the induction, we added chloramphenicol into the medium and measured the optical density for about 10 hours to estimate the concentration of the cell.

Without induction of C4HSL, the cell cannot express CmR resistance gene and cannot survive in the presence of chloramphenicol. As shown in Fig. 2-1-11, when C4HSL is added to the culture, Company cell survived and increased. This result indicates that Company cell produced 3OC12HSL in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI. From these experiments, we confirmed that a new part Prhl(RL)-CmR-LasI synthesized CmR and 3OC12HSL work as expected.

 
Fig. 2-1-11. Company cannot grow without C4HSL
 

To characterize the function of C4HSL-dependent 3OC12HSL production, we also performed a reporter assay by using lux reporter cell (Fig. 2-1-12) First, the expression of LasI was induced by adding C4HSL to the culture of the Company cell. Then, the supernatant of the culture was added to the culture of reporter cell. The expression of GFP in the reporter cell was measured by flow cytometer.

 

As Fig. 2-1-12 shows, when the reporter cell E was incubated in the culture of the induced Company cell, the fluorescence intensity of the reporter cell increased. Comparing the results of the culture with the induced Company cell and not induced Company cell, the reporter cell in the supernatant of induced cell had 29-fold higher fluorescence intensity.

This result indicates that Company cell produced 3OC12HSL in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI. From these experiment, we confirmed that a new part Prhl(RL)-CmR-LasI synthesized CmR and 3OC12HSL as expected.

Fig. 2-1-12. Company excretes 3OC12HSL by new BioBrick parts when C4HSL exists
 
2.4.2. Customer
 

For construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL production module, we designed a new part Plux-CmR-RhlI (BBa_K1529797)(Fig. 2-1-13). In order to confirm the Customer’s dependency on 3OC12HSL, we measured the growth of Customer cell in the presence and absence of 3OC12HSL. After induction, we added chloramphenicol into the medium and measured optical density for about ten hours to estimate the concentration of the cell.

Without induction of 3OC12HSL, the cell cannot express CmR and cannot survive in the presence of chloramphenicol. As shown in Fig. 2-1-14, when 3OC12HSL was added to the culture, Customer cell survived and increased. This result indicates that CmR was produced in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.

Fig. 2-1-13. Genetic circuit design of Customer
 
 
Fig. 2-1-14. Customer cannot survive without 3OC12HSL
 

To characterize the function of 3OC12HSL-dependent C4HSL production, we also performed a reporter assay by using lux reporter cell (Fig. 2-1-15).

First, the expression of RhlI was induced by adding 3OC12HSL to the culture of the Customer cell. Then, the supernatant of the culture was added to the culture of reporter cell. The expression of GFP in the reporter cell was measured by flow cytometer.

As Fig. 2-1-15 shows, when the reporter cell Plux-CmR-RhlI was incubated in the culture of the induced Customer cell, the fluorescence intensity of the reporter cell increased. Comparing the results of the induced cell and not induced dell, reporter cell in the supernatant of the induced cell had 95-fold higher fluorescence intensity. This result indicates that Customer cell produced C4HSL in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.

From these experiments, we confirmed that a new part Plux-CmR-RhlI synthesized CmR and C4HSL as expected.

 

Fig. 2-1-15. Customer excretes C4HSL when C12HSL exists
 

2.5. Assay of symbiosis between Company and Customer by co-culture

For the accomplishment of mutualism between the Company cells and Customer cells, we mixed and co-cultured the two cells to show symbiosis of them. Company’s characteristics are C4HSL-dependent survival and 3OC12HSL production, and Customer’s characteristics are the opposite from Company’s. (If you want to know about these cells in more detail, see the above section. Each cells’ function is described.) From these characteristics, the symbiosis between the two cells can be established.

 
Fig. 2-1-16. The result of co-culture assay
 

Two types of fluorescent proteins were used to trace the growth of each cells in our symbiosis experiments. We constructed the Company cell containing GFP and Customer cell containing RFP. By measuring the OD of the cells expressing GFP with flow cytometer, the symbiosis was detected.

The result of the co-culture assay is shown in Fig. 2-1-16. By looking at the fluorescence intensity of GFP in Company cells, the optical density increased faster in co-culture than single culture. From this point, we can say that Company and Customer actually mutualize in the medium.

 
 
 

3. Addition of Bank

We have demonstrated the mutualism between Company and Customer through modeling and wet-experiments. However, thousands of Company-Customer pairs exists in the actual economy. In the experimental design of such next demonstration, our modeling suggests that our educational tool of economics include an additional player, Bank.

 

3.1. Motivation

In the experimental design to expand the number of Company-Customer pairs in one test tube, we found that carrying capacity of a medium is shared among the pairs. In other words, the amount of cell corresponding to each pair must be less to establish the symbiosis.

Our modeling, however, suggested that a certain amount of cells is needed to maintain the symbiosis between Company and Customer (See 2.2. Condition for the optimal growth deducted from simulation).

We noticed that the introduction of bank may assist not only the symbiosis with small amount of cells in a test tube, but also the understanding of economics as an education tool. In real economy, a central bank will supply money to the market when money is in short supply. Therefore, we designed Bank E. coli that does the same work as central bank.

 

3.2. Genetic circuit design of Bank E. coli

Fig. 2-1-17. Genetic circuit of Bank E. coli
 

The circuit design of Bank E. coli is shown in Fig. 2-1-16 Bank E. coli functions as a central bank to regulate the money supply in the market. Bank E. coli can change into two states by using toggle switch (Gardner, 2000), which depends on C4HSL concentration (Fig. 2-1-17).

  The whole mechanism this state switching is explained below in four steps.

 
3.2.1 Distribution state

When Bank is in the distribution state, RhlI in Bank will be expressed to produce C4HSL, which is the money supplied to the market.

Fig. 2-1-18. Distribution state
 
3.2.2 Change to Collection State

If Bank is always in the distribution state, too much money will be supplied to the economy. Thus when the amount of C4HSL is excessed which will activate Prhl/lac promoter to express AiiA. When Bank finishes this change, it will switch to the collection state.

Fig. 2-1-19. Change to collection state
 
3.2.3 Collection State

If the amount of money in the economy becomes excessive, Bank will be in the collection state. In this state, AiiA in Bank will be expressed. AiiA decomposes C4HSL and 3OC12HSL in the medium to decrease the money supply in the economy.

Fig. 2-1-20. Collection State
 
3.2.4 Change to distribution state

For maintenance of symbiosis even in lack of money supply, Bank changes to the distribution state. In this step, transcription in Prhl/lac promoter is stopped by lower C4HSL concentration resolved by AiiA. Therefore, TetR will lower the expression. Instead, Ptet promoter is activated to express RhlI and LacI.

Fig. 2-1-21. Change to distribution state
 

3.3. Modeling: Bank actually helps the development of the economy

We made a mathematical model to ensure the functioning of the Bank. Bank helps Company and Customer to grow well even with a few amounts. As shown in Fig. 2-1-22, if the amounts of Company and Customer are few, then they cannot grow well. But once the Bank is included, all of them can grow well.

 
Fig. 2-1-22. Bank helps Company and Customer to grow well
 

Also to ensure the functioning, we analyzed the Bank’s internal switch depending on the C4HSL concentration. Fig. 2-1-23 shows the RhlI concentration depending on the C4 concentration in the Bank cell. When the C4 concentration increases from low to high, the RhlI concentration follows the green line on the figure, decreasing the RhlI expression. This means the Bank switches its state from distribution state to collection state. On the other hand, when the C4 concentration decreases, the RhlI concentration follows the blue line, increasing the RhlI expression. This means the Bank switches its state from collection state to distribution state.

 
Fig. 2-1-23. Bank changes its state from Distribution State to Collection State
 

"These results show that Bank functions as a central bank as in the real economy."

 
 
 

4. Economic Wave: Advice from Entrepreneurs

We introduced our Bank E. coli project to businessmen, executives, and technicians engaged in IT companies at a workshop called MUSE TALK for professional opinions (See Policy and Practice for more details). They pointed out that economic wave should be integrated into our system to make it more realistic (Fig. 2-1-24).

 
Fig. 2-1-24. Our teammates exchanging opinions with professionals
 

In the real economy, there are long-term financial wave named Kitchin inventory, Kondratiev wave etc. (Wikipedia.org). These long-term financial waves deeply affect the whole economy. We were advised that ignoring these waves can be a crucial defect in our project.

In order to develop our educational tool for iGEMERs more, we merged the idea of economic wave into our system as the fluctuation of C4HSL concentration. Even though Company and Customer can grow well only by themselves as shown in Fig. 2-1-25(left), they cannot endure the effect of economic wave as shown in Fig. 2-1-25(center). These results show that Company and Customer are not good at dealing with the economic wave. Let us introduce the Bank to the market with economic wave. As shown in Fig. 2-1-25(right), Bank helps Company and Customer to grow well.

 
Fig. 2-1-25. Company and Customer cannot survive when they face economic wave.
 

Money supplies in these situations, represented by C4 concentrations, are shown in Fig. 2-1-26. Even though the money supply decreases very much by the effect of economic wave as shown in Fig,2-1-26(center), the introduction of the Bank changes the state drastically, results in abundant money supply Fig. 2-1-26(right).

 
Fig. 2-1-26 Money supply (C4 concentration) in every situation described in Fig. 2-1-25.
 

Although the above results show how Bank stabilizes the system, small change of parameter in economic wave makes Bank to fail managing. With harsh economic wave, the effect of economic wave begins to be out of hands of Bank. As shown in Fig.2-1-27(left), the population of Company and Customer fluctuates very much. This is due to the fluctuation of money supply in the market(Fig. 2-1-27(right)). Interestingly, the Bank constantly grows while the other two suffers the fluctuation of the money supply (Fig. 2-1-27(left)). Be careful not to be like this in your countries.

 
Fig. 2-1-27. Bank can help Company and Customer even with harsh economic wave
 
 
 

5. Project Evaluation

 

Finally, the evaluation of our educational tool using E. coli and BioBrick showed that our project is highly effective for students majoring in biology to understand economics (Fig. 2-1-28).

 
Fig. 2-1-28. Effects of our presentation
 
 
 
 

6. Reference

 
1. Frederick K Balagadde et al. (2008) A synthetic Escherichia coli predator–prey ecosystem. Molecular Systems Biology 4: 187
2. Alissa Kerner et al. (2012) A Programmable Escherichia coli Consortium via Tunable Symbiosis. PLoS ONE 7(3): e34032
3. Jennifer M. Henke et al. (2004) Bacterial social engagements. TRENDS in Cell Biology 14: 11
4. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619
5. John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. SCIENCE 323: 272-275
6. Hideki Kobayashi et al. (2004) Programmable cells: Interfacing natural and engineered gene networks. PNAS 101: 8414-8419
7. Lingchong You et al. (2004) Programmed population control by cell–cell communication and regulated killing. NATURE 428: 868-871
8. Timothy S. Gardner et al. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339-342
9. Roji Sekine et al. (2011) Tunable synthetic phenotypic diversification on Waddington’s landscape through autonomous signaling. PNAS 108: 17969-17973
10. Kendall M. Gray et al. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology 176(10): 3076–3080