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| <h3 class="link"><a href="#sce4"><span style="font-size: 14px;"> 4.4. Scene4 : The Prince's rescue</span></a></h3> | | <h3 class="link"><a href="#sce4"><span style="font-size: 14px;"> 4.4. Scene4 : The Prince's rescue</span></a></h3> |
| <h3 class="link"><a href="#Q.E.D.">5. Q.E.D. for the representation of Snow White</a></h3> | | <h3 class="link"><a href="#Q.E.D.">5. Q.E.D. for the representation of Snow White</a></h3> |
− | <h3 class="link"><a href="#5-sce1"><span style="font-size: 14px;"> 5.1. Cold inducible promoter functions at 18°C in Scene 1</span></a></h3> | + | <h3 class="link"><a href="#sysscene1"><span style="font-size: 14px;"> 5.1. Cold inducible promoter functions at 18°C in Scene 1</span></a></h3> |
− | <h3 class="link"><a href="#5-sce2"><span style="font-size: 14px;"> 5.2. The screening of rhl promoter with optimal strength for Scene 2</span></a></h3> | + | <h3 class="link"><a href="#sysscene2"><span style="font-size: 14px;"> 5.2. The screening of rhl promoter with optimal strength for Scene 2</span></a></h3> |
| <h3 class="link"><a href="#ass"><span style="font-size: 14px;"> 5.2.1. reporter assay</span></a></h3> | | <h3 class="link"><a href="#ass"><span style="font-size: 14px;"> 5.2.1. reporter assay</span></a></h3> |
| <h3 class="link"><a href="#sml"><span style="font-size: 14px;"> 5.2.2. Simulation regarding Prhl strength</span></a></h3> | | <h3 class="link"><a href="#sml"><span style="font-size: 14px;"> 5.2.2. Simulation regarding Prhl strength</span></a></h3> |
| <h3 class="link"><a href="#imp"><span style="font-size: 14px;"> 5.2.3. Improvement of Prhl</span></a></h3> | | <h3 class="link"><a href="#imp"><span style="font-size: 14px;"> 5.2.3. Improvement of Prhl</span></a></h3> |
− | <h3 class="link"><a href="#5-sce3"><span style="font-size: 14px;"> 5.3. MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3</span></a></h3> | + | <h3 class="link"><a href="#sysscene3"><span style="font-size: 14px;"> 5.3. MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3</span></a></h3> |
− | <h3 class="link"><a href="#5-sce4"><span style="font-size: 14px;"> 5.4. AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene4</span></a></h3> | + | <h3 class="link"><a href="#sysscene4"><span style="font-size: 14px;"> 5.4. AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene4</span></a></h3> |
| <h3 class="link"><a href="#5-sml"><span style="font-size: 14px;"> 5.5. the simulation related to the story</span></a></h3> | | <h3 class="link"><a href="#5-sml"><span style="font-size: 14px;"> 5.5. the simulation related to the story</span></a></h3> |
| <h3 class="link"><a href="#5-str"><span style="font-size: 14px;"> 5.5.1. When the Prince comes?</span></a></h3> | | <h3 class="link"><a href="#5-str"><span style="font-size: 14px;"> 5.5.1. When the Prince comes?</span></a></h3> |
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| </div><!-- /_header --> | | </div><!-- /_header --> |
| <div id="introduction_contents" class="container_contents"> | | <div id="introduction_contents" class="container_contents"> |
− | <p class="normal_text">When you were little, before going to sleep, you have probably asked your mother, "Mommy, read some picture books to me, please?" Your mother may have read aloud many stories. </p> | + | <p class="normal_text">When you were little, before going to sleep, you have probably asked your mother, "Mommy, read some picture books to me, please?" Your mother may have read aloud many stories. Most of the stories that have been told for generation contain lessons in them. The lessons from the Snow White story are mainly the following two points.</p> |
− | <p class="normal_text">Most of the stories that have been told for generation contain lessons in them. The lessons from the Snow White story are mainly the following two points.</p>
| + | |
| <p class="normal_text">Ⅰ. Do not show off or envy someone.</p> | | <p class="normal_text">Ⅰ. Do not show off or envy someone.</p> |
| <p class="normal_text">Ⅱ. Do not trust unfamiliar people blindly. </p> | | <p class="normal_text">Ⅱ. Do not trust unfamiliar people blindly. </p> |
− | <p class="normal_text">For a beautiful mind, we need not only to be kind and generous but also to keep a strong heart and control ourselves.</p>
| + | <p class="normal_text"> For a beautiful mind, we need not only to be kind and generous but also to keep a strong heart and control ourselves.In our project, the Snow White story is going to be recreated and Snow White and the Queen is going to be evaluated in terms of the beauty from both inside and outside.</p> |
− | <p class="normal_text">In our project, the Snow White story is going to be recreated and Snow White and the Queen is going to be evaluated in terms of the beauty from both inside and outside.</p>
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| <div id="stories"> | | <div id="stories"> |
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− | <div align="center"><span style="font-style : italic; font-size:18px;">"Magic Mirror on the wall, who is the fairest one of all?"</span></div> | + | <div align="center"><span style="font-style : normal_text; font-size:18px;">"Magic Mirror on the wall, who is the fairest one of all?"</span></div><br><br> |
− | <p class="normal_text">Once upon a time, there lived a Queen.<br> | + | <p class="normal_text">Once upon a time, there lived a Queen.</p><br> |
− | She was the fairest in the world and she herself also believed so.<br><br> | + | <p class="normal_text">She was the fairest in the world and she herself also believed so.</p><br><br> |
− | Each time the Queen asked, "Magic Mirror on the wall, who is the fairest one of all?," the mirror would give the same answer.<br> | + | <p class="normal_text">Each time the Queen asked, </p><br> |
− | "You are the fairest one of all."<br> | + | <p class="normal_text">"Magic Mirror on the wall, who is the fairest one of all?," the mirror would give the same answer.</p><br> |
− | This pleased the Queen greatly, for she knew that her Magic mirror could speak nothing but the truth and she asked it the same question.<br><br> | + | <p class="normal_text">"You are the fairest one of all."</p><br><br> |
− | "Magic Mirror on the wall, who is the fairest one of all?"<br><br> | + | <p class="normal_text">This pleased the Queen greatly, for she knew that her Magic mirror could speak nothing but the truth and she asked it the same question.</p><br><br> |
− | One winter night, the Queen asked her mirror as usual.<br> | + | <p class="normal_text">"Magic Mirror on the wall, who is the fairest one of all?"</p><br><br> |
− | "Magic mirror on the wall, who is the fairest one of all?"<br> | + | <p class="normal_text">One winter night, the Queen asked her mirror as usual.</p><br> |
− | The mirror answered,<br> | + | <p class="normal_text">"Magic mirror on the wall, who is the fairest one of all?"</p><br> |
− | "Snow White is the fairest one."</p><br><br> | + | <p class="normal_text">The mirror answered,</p><br> |
| + | <p class="normal_text">"Snow White is the fairest one."</p><br><br> |
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| <p class="normal_text">The Snow White was Queen's daughter‐in‐law.</p> | | <p class="normal_text">The Snow White was Queen's daughter‐in‐law.</p> |
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− | <p class="normal_text">THE END</p><br><br><br><br> | + | <p class="normal_text">THE END</p><br><br> |
− | | + | |
| <div class="gradation_cover"><a href="javascript:void(0);" onclick="show('stories'); return false;"><img src="https://static.igem.org/mediawiki/2016/5/54/T--Tokyo_Tech--readmore.png" /></a></div><!--- /.gradation_cover --> | | <div class="gradation_cover"><a href="javascript:void(0);" onclick="show('stories'); return false;"><img src="https://static.igem.org/mediawiki/2016/5/54/T--Tokyo_Tech--readmore.png" /></a></div><!--- /.gradation_cover --> |
| </div><!-- /stories --> | | </div><!-- /stories --> |
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| <p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Real Beauty = outer beauty + inner beauty </span> | | <p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Real Beauty = outer beauty + inner beauty </span> |
− | <p class="normal_text">To evaluate the Real beauty, we are going to recreate the Snow White story using E.coli.</p> | + | <p class="normal_text">To evaluate the Real beauty, we are going to recreate the Snow White story using <span style="font-style:italic;">E. coli</span>.</p> |
| </p><br> | | </p><br> |
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| </p></div><br> | | </p></div><br> |
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− | <p class="normal_text"> To evaluate the real beauty, we recreated Snow White story by using E.coli.<br> | + | <p class="normal_text"> To evaluate the real beauty, we recreated Snow White story by using <span style="font-style:italic;">E.coli</span>.<br> |
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| </p> | | </p> |
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| <div style="text-align:center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png"><img src="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png" width="600px" /></a></div> | | <div style="text-align:center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png"><img src="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png" width="600px" /></a></div> |
| <p class="normal_text">The basic design of Snow White <span style="font-style: italic;">coli</span>'s genetic circuit is almost the same as the Queen <span style="font-style: italic;">coli</span>'. | | <p class="normal_text">The basic design of Snow White <span style="font-style: italic;">coli</span>'s genetic circuit is almost the same as the Queen <span style="font-style: italic;">coli</span>'. |
− | Snow White coli receives 3OC12HSL, the Poisoned Apple, and expresses RhlI and MazF. RhlI synthesizes signaling molecule C4HSL. As described in the previous section, the Queen <span style="font-style: italic;">coli</span> receives C4HSL and monitor Snow White coli through it. MazF, on the other hand, inhibits the translation in Snow White <span style="font-style: italic;">coli</span>. | + | Snow White <span style="font-style:italic;">coli</span> receives 3OC12HSL, the Poisoned Apple, and expresses RhlI and MazF. RhlI synthesizes signaling molecule C4HSL. As described in the previous section, the Queen <span style="font-style: italic;">coli</span> receives C4HSL and monitor Snow White<span style="font-style:italic;"> coli</span> through it. MazF, on the other hand, inhibits the translation in Snow White <span style="font-style: italic;">coli</span>. |
| <br> | | <br> |
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| <div style="text-align: center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png"height="100"><img src="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png" width="400px" /></a></div> | | <div style="text-align: center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png"height="100"><img src="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png" width="400px" /></a></div> |
− | <p class="normal_text">The Prince coli which receives 3OC12HSL expresses AmiE. AmiE is said to degrade HSL with more than 6 carbons, which means that AmiE degrades the Poisoned Apple, 3OC12HSL.By the degradation of the Poisoned Apple, MazF expression is inhibited in Snow White and its function is counteracted by MazE. Then, translation restarts in Snow White <span style="font-style: italic;">coli</span>.</p> | + | <p class="normal_text">The Prince <span style="font-style:italic;">coli</span> which receives 3OC12HSL expresses AmiE. AmiE is said to degrade HSL with more than 6 carbons, which means that AmiE degrades the Poisoned Apple, 3OC12HSL.By the degradation of the Poisoned Apple, MazF expression is inhibited in Snow White and its function is counteracted by MazE. Then, translation restarts in Snow White <span style="font-style: italic;">coli</span>.</p> |
| </div> | | </div> |
| </div> | | </div> |
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| <h3><span>5.1 Cold inducible promoter functions at 18°C in Scene 1</span></h3> | | <h3><span>5.1 Cold inducible promoter functions at 18°C in Scene 1</span></h3> |
| </div><!-- /_header --> | | </div><!-- /_header --> |
− | <div id="sys1"> | + | <div id="sysscene1"> |
− | <div id="sys1_contents"> | + | <div id="sysscene1_contents"> |
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| <div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f5/T--Tokyo_Tech--koma1.png" height ="200"></div><br> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f5/T--Tokyo_Tech--koma1.png" height ="200"></div><br> |
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− | <p class="normal_text">5.2 The screening of Prhl with optimal strength for Scene 2<br>
| + | <h3><span>5.2 The screening of Prhl with optimal strength for Scene 2</span><h3> |
| + | </div><!-- /_header --> |
| + | <div id="sysscene2"> |
| + | <div id="sysscene2_contents"> |
| + | |
| + | |
| <div align="center"><a href="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"></a><br></div> | | <div align="center"><a href="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"></a><br></div> |
| <p class="normal_text">Eating the Poisoned Apple given by the Queen <span style="font-style : italic">coli</span>, the Snow White <span style="font-style : italic">coli</span> falls asleep. We exploit the cell-cell communication mechanism of bacteria to represent this scene, and so we need promoters with optimal strength. Otherwise, our final genetic circuit never work properly.</p> | | <p class="normal_text">Eating the Poisoned Apple given by the Queen <span style="font-style : italic">coli</span>, the Snow White <span style="font-style : italic">coli</span> falls asleep. We exploit the cell-cell communication mechanism of bacteria to represent this scene, and so we need promoters with optimal strength. Otherwise, our final genetic circuit never work properly.</p> |
| <p class="normal_text">Therefore, we introduce rhl system assay as a concept in this Scene. We improved the function of Prhl through the process listed below and succeeded in newly obtaining of optimal Prhl mutants for our project. </p> | | <p class="normal_text">Therefore, we introduce rhl system assay as a concept in this Scene. We improved the function of Prhl through the process listed below and succeeded in newly obtaining of optimal Prhl mutants for our project. </p> |
| <p class="normal_text"> 5.2.1 Reporter assay<br> | | <p class="normal_text"> 5.2.1 Reporter assay<br> |
| + | </div><!-- /_header --> |
| + | <div id="ass"> |
| + | <div id="ass_contents"> |
| <p class="normal_text">First, we evaluated the activities the existing promoters, Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) by observing the reactivity to three AHLs (= expression inducers, C4HSL, 3OC6HSL, and 3OC12HSL). | | <p class="normal_text">First, we evaluated the activities the existing promoters, Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) by observing the reactivity to three AHLs (= expression inducers, C4HSL, 3OC6HSL, and 3OC12HSL). |
| </p> | | </p> |
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| <p class="normal_text"> 5.2.2 Simulation regarding Prhl strength <br> | | <p class="normal_text"> 5.2.2 Simulation regarding Prhl strength <br> |
| + | </div><!-- /_header --> |
| + | <div id="sml"> |
| + | <div id="sml_contents"> |
| <p class="normal_text"> Based on the experimental results in the preceding paragraph, we performed a simulation to analyze whether our genetic circuit works properly when the existing Prhl was used.<br> | | <p class="normal_text"> Based on the experimental results in the preceding paragraph, we performed a simulation to analyze whether our genetic circuit works properly when the existing Prhl was used.<br> |
| + | <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/68/T--Tokyo_Tech--projct_model1.png" height ="300"><br></div> |
| + | |
| <p class="normal_text">The above diagram shows the intensity of the two promoters should be in the red region in the figure. The green point shows the relationships of the promoters which we intended to use primarily. In order to move to the red region, we found that it is needed to improve Prhl and increase its expression level.</p> | | <p class="normal_text">The above diagram shows the intensity of the two promoters should be in the red region in the figure. The green point shows the relationships of the promoters which we intended to use primarily. In order to move to the red region, we found that it is needed to improve Prhl and increase its expression level.</p> |
− | <p class="normal_text">The graph below shows the result of recreating “Snow White” when using existing rhl promoter. Only the fluorescence intensity of the Queen coli got lowered. It means that the Queen coli failed to poison Snow White coli and she killed herself.</p>
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− | <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/68/T--Tokyo_Tech--projct_model1.png" height ="300"><br></div>
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− | <div align="center"> | + | |
| <a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prhl ">Read Model page</a>. | | <a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prhl ">Read Model page</a>. |
| </p><br> | | </p><br> |
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| <p class="normal_text"> 5.2.3 Improvement of Prhl<br> | | <p class="normal_text"> 5.2.3 Improvement of Prhl<br> |
| + | </div><!-- /_header --> |
| + | <div id="imp"> |
| + | <div id="imp_contents"> |
| We generated mutants of Prhl by introducing a point mutation into the wild type (WT) Prhl. In the experiment, we added reagent AHLs into the reporter cells and examined their fluorescence intensities. As a result, we succeeded in newly obtaining stronger mutants than the WT Prhl. The new mutant was named Prhl (NM).<br> | | We generated mutants of Prhl by introducing a point mutation into the wild type (WT) Prhl. In the experiment, we added reagent AHLs into the reporter cells and examined their fluorescence intensities. As a result, we succeeded in newly obtaining stronger mutants than the WT Prhl. The new mutant was named Prhl (NM).<br> |
| <div align="center"><img src="https://static.igem.org/mediawiki/2016/e/ef/Tokyo_Tech_Prhlimp.png" height ="300"><br></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/e/ef/Tokyo_Tech_Prhlimp.png" height ="300"><br></div> |
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| </p><br> | | </p><br> |
− | <div id="sce2_header">
| + | |
| <h3><span>5.3 MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3</span></h3> | | <h3><span>5.3 MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3</span></h3> |
| </div><!-- /_header --> | | </div><!-- /_header --> |
| + | <div id="sysscene3"> |
| + | <div id="sysscene3_contents"> |
| + | |
| <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Tokyo_Tech--koma3.png" height ="200"></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Tokyo_Tech--koma3.png" height ="200"></div> |
| <p class="normal_text">In this Scene, we introduce TA system as a concept.</p> | | <p class="normal_text">In this Scene, we introduce TA system as a concept.</p> |
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| <p class="normal_text">According to the experiments, we showed that TA system works properly.</p> <a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay/mazEF_System_Assay | | <p class="normal_text">According to the experiments, we showed that TA system works properly.</p> <a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay/mazEF_System_Assay |
| ">Read mazEF System Assay</a>. | | ">Read mazEF System Assay</a>. |
| + | |
| </p><br> | | </p><br> |
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− | <p class="normal_text">5.4 AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene 4<br>
| + | <h3><span>5.4 AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene 4</span></h3></span> |
| + | </div><!-- /_header --> |
| + | <div id="sysscene4"> |
| + | <div id="sysscene4_contents"> |
| + | |
| <div align="center"><img src="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png" height ="200"></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png" height ="200"></div> |
| <p class="normal_text">In the final Scene, we introduce the selective degradation of AHLs by AmiE as a concept. | | <p class="normal_text">In the final Scene, we introduce the selective degradation of AHLs by AmiE as a concept. |
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| </p><br> | | </p><br> |
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− | <p class="normal_text">5.5 the simulation related to the story<br>
| + | <h3><span>5.5 the simulation related to the story</span></h3> |
| + | </div><!-- /_header --> |
| + | <div id="5-sml"> |
| + | <div id="5-sml_contents"> |
| + | <p class="normal_text"> |
| + | |
| From the results of wet lab, it was showed that the designed circuit works properly at each point. We simulated the representation of the story in a combination of these Points.<br> | | From the results of wet lab, it was showed that the designed circuit works properly at each point. We simulated the representation of the story in a combination of these Points.<br> |
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| <p class="normal_text">From this result, it was found that the genetic circuit works well by adding the Prince <span style="font-style : italic">coli</span> at t=700. In other words, as with the original story, the Prince <span style="font-style : italic">coli</span> comes across Snow White <span style="font-style : italic">coli</span> at t=700 and rescues her.<br></p><a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prince_coli ">Read Model page</a>. | | <p class="normal_text">From this result, it was found that the genetic circuit works well by adding the Prince <span style="font-style : italic">coli</span> at t=700. In other words, as with the original story, the Prince <span style="font-style : italic">coli</span> comes across Snow White <span style="font-style : italic">coli</span> at t=700 and rescues her.<br></p><a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prince_coli ">Read Model page</a>. |
| + | </div><!-- /_header --> |
| + | <div id="5-str"> |
| + | <div id="5-str_contents"> |
| + | |
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| </p><br> | | </p><br> |
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| <p class="normal_text">5.5.2 representation of the story<br> | | <p class="normal_text">5.5.2 representation of the story<br> |
| + | </div><!-- /_header --> |
| + | <div id="5-str2"> |
| + | <div id="5-str2_contents"> |
| As a result of simulation, we obtained and confirmed the desirable behavior of the whole system by modifying and improving parts. As described below, our simulation showed appropriate transition of fluorescence for the story.<br> | | As a result of simulation, we obtained and confirmed the desirable behavior of the whole system by modifying and improving parts. As described below, our simulation showed appropriate transition of fluorescence for the story.<br> |
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| In the yellow area of Fig.5-2-2, the AmiE synthesized by the introduced Prince <span style ="font-style : italic">coli</span> decomposes C12 so the MazF inside Snow White <span style ="font-style : italic">coli</span> diminishes and the concentration of GFP resumes. <br> | | In the yellow area of Fig.5-2-2, the AmiE synthesized by the introduced Prince <span style ="font-style : italic">coli</span> decomposes C12 so the MazF inside Snow White <span style ="font-style : italic">coli</span> diminishes and the concentration of GFP resumes. <br> |
− | It looks as if the Prince lifted Snow White and she opened her eyes.</p><a href="https://2016.igem.org/Team:Tokyo_Tech/Model#results">Read Model page</a>. | + | It looks as if the Prince lifted Snow White and she opened her eyes. |
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| Then, our dry lab used Java to develop software named “ACA Dwarfs” adjusting number of ACA base sequence as a solution to the problem.</p> | | Then, our dry lab used Java to develop software named “ACA Dwarfs” adjusting number of ACA base sequence as a solution to the problem.</p> |
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− | <p class="normal_text">This graph shows that the simulation result when changing the base sequences of GFP and RFP with ACA Dwarfs. In case of GFP and RFP, their concentrations decreases earlier than original ones.<br>
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− | Using ACADwarfs enabled us to make GFP and RFP more reactive to MazF.<br></p>
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