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| “Snow White is the fairest one.” </span><br><br> | | “Snow White is the fairest one.” </span><br><br> |
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− | The magic Mirror coli can produce RhlI protein under low temperature. Rhl protein production leads to production of the signaling molecule, C4HSL which tells the Queen that Snow White is the fairest,and the Queen coli receives this molecule. | + | The magic Mirror <span style="font-style: italic">coli</span> can produce RhlI protein under low temperature. Rhl protein production leads to production of the signaling molecule, C4HSL which tells the Queen that Snow White is the fairest,and the Queen <span style="font-style: italic">coli</span> receives this molecule. |
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| </p> | | </p> |
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| <p class="normal_text"><span style="font-style : italic">Snow White, who always takes people at their word, bit the apple, then sank into unconsciousness soon.</span><br><br> | | <p class="normal_text"><span style="font-style : italic">Snow White, who always takes people at their word, bit the apple, then sank into unconsciousness soon.</span><br><br> |
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− | 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> s.<br> | + | 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> s.<br> |
| Snow White <span style="font-style : italic">coli</span>, which has received the C12HSL, the Poisoned Apple, expresses <span style="font-style : italic">rhlI</span> and MazF.<br> | | Snow White <span style="font-style : italic">coli</span>, which has received the C12HSL, the Poisoned Apple, expresses <span style="font-style : italic">rhlI</span> and MazF.<br> |
− | Produced RhlI produces a signaling molecule: C4HSL. As was described in the introduction of the Queen in the previous section , it can receive C4HSL and monitor Snow White coli through this.<br> | + | Produced RhlI produces a signaling molecule: C4HSL. As was described in the introduction of the Queen in the previous section , it can receive C4HSL and monitor Snow White <span style="font-style: italic">coli</span> through this.<br> |
| In addition, expressed MazF stops the translation in the Snow White <span style="font-style : italic">coli</span>. | | In addition, expressed MazF stops the translation in the Snow White <span style="font-style : italic">coli</span>. |
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| </span><br><br> | | </span><br><br> |
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− | The Prince coli has received 3OC12HSL expresses AmiE. In general, it is said that AmiE degrades HSL with C8 or more. This leads to degradation of the Poisoned Apple that is 3OC12HSL.<br> | + | The Prince <span style="font-style: italic">coli</span> has received 3OC12HSL expresses AmiE. In general, it is said that AmiE degrades HSL with C8 or more. This leads to degradation of the Poisoned Apple that is 3OC12HSL.<br> |
− | When the poison apple is degraded, MazF expression stops in Snow White, and gradually its function is canceled by MazE. Then, translation restarts in Snow White coli. | + | When the poison apple is degraded, MazF expression stops in Snow White, and gradually its function is canceled by MazE. Then, translation restarts in Snow White <span style="font-style: italic">coli</span>. |
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| </p> | | </p> |
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| <div id="fun2_contents"> | | <div id="fun2_contents"> |
| <div align="center"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"><br></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"><br></div> |
− | <p class="normal_text">Snow White coli dies by the Poisoned Apple given by the Queen . The reporter with optimal strength is needed to work the genetic circuit which we designed properly. For this reason, we introduce rhl system assay as a concept in this Scene. We improved the function of rhl promoter through the process listed below and succeeded in newly getting of rhl promoter mutants optimal for our project.<br><br> | + | <p class="normal_text">Snow White <span style="font-style: italic">coli</span> dies by the Poisoned Apple given by the Queen . The reporter with optimal strength is needed to work the genetic circuit which we designed properly. For this reason, we introduce rhl system assay as a concept in this Scene. We improved the function of rhl promoter through the process listed below and succeeded in newly getting of rhl promoter mutants optimal for our project.<br><br> |
| 4.2.1 reporter assay<br> | | 4.2.1 reporter assay<br> |
| First of all, we evaluated the activities the existing promoters: Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) when added 3 types of AHLs. | | First of all, we evaluated the activities the existing promoters: Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) when added 3 types of AHLs. |
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| <p class="normal_text">In this experiment, we used the BBa_K1949100 and Bba_1949102.<br> | | <p class="normal_text">In this experiment, we used the BBa_K1949100 and Bba_1949102.<br> |
| First, MazF was expressed by arabinose, and 2 h later, the MazE was expressed by IPTG.<br> | | First, MazF was expressed by arabinose, and 2 h later, the MazE was expressed by IPTG.<br> |
− | From the experimental results, we found that the turbidity of samples without MazE turbility did not rise. However, we also found that E. coli restarts its cell growth when MazE is expressed by adding IPTG.<br> | + | From the experimental results, we found that the turbidity of samples without MazE turbility did not rise. However, we also found that <span style="font-style: italic">E. coli</span> restarts its cell growth when MazE is expressed by adding IPTG.<br> |
| Moreover, when only MazF worksed, the RFU of GFP hardly rose, but when MazE was induced, the RFU of GFP rose. (link: toxin assay)<br> | | Moreover, when only MazF worksed, the RFU of GFP hardly rose, but when MazE was induced, the RFU of GFP rose. (link: toxin assay)<br> |
− | From the above, we concluded that MazF stops cell growth and translation of E. coli, but MazE restarts cell growth that have stopped and furthermore restarts translation.<br> | + | From the above, we concluded that MazF stops cell growth and translation of <span style="font-style: italic">E. coli</span>, but MazE restarts cell growth that have stopped and furthermore restarts translation.<br> |
| According to the experiments, we showed that TA system works properly. | | According to the experiments, we showed that TA system works properly. |
| </p> | | </p> |
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| </p><br> | | </p><br> |
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− | <p class="normal_text">From the experimental results, when C12 was added to the culture skution of E. coli where AmiE was expressed, C12 was degraded, whereas C4 was hardly degraded.<br> | + | <p class="normal_text">From the experimental results, when C12 was added to the culture skution of <span style="font-style: italic">E. coli</span> where AmiE was expressed, C12 was degraded, whereas C4 was hardly degraded.<br> |
| For these reasons, we showed that AmiE selectively degrades AHLs, only C12 in this project. | | For these reasons, we showed that AmiE selectively degrades AHLs, only C12 in this project. |
| </p> | | </p> |
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| 4.5.1 王子様いつやってくのだろうか<br> | | 4.5.1 王子様いつやってくのだろうか<br> |
− | We simulated to confirm which is better, adding the Prince coli first or adding it in midstream, to make our genetic circuit work. <br> | + | We simulated to confirm which is better, adding the Prince <span style="font-style: italic">coli</span> first or adding it in midstream, to make our genetic circuit work. <br> |
− | As a result, we found that when the Prince coli is added from the beginning, AmiE produced by the Prince coli increases and C12 is degraded too much. Then, C12 cannot exist in the medium (Fig. 2) and the circuit does not work correctly. | + | As a result, we found that when the Prince <span style="font-style: italic">coli</span> is added from the beginning, AmiE produced by the Prince <span style="font-style: italic">coli</span> increases and C12 is degraded too much. Then, C12 cannot exist in the medium (Fig. 2) and the circuit does not work correctly. |
| </p><br> | | </p><br> |
| <div align="center"><img src="https://static.igem.org/mediawiki/2016/1/1a/T--Tokyo_Tech--project_model1.png" height ="300"><br></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/1/1a/T--Tokyo_Tech--project_model1.png" height ="300"><br></div> |
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| </p></div><br> | | </p></div><br> |
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− | <p class="normal_text">On the other hand, when the Prince coli is added at t = 700, the number of the Prince coli does not increase greatly (Fig3). Therefore, C12 can exist until t = 70, and after that C12 decreases with an increase in AmiE (Fig4). | + | <p class="normal_text">On the other hand, when the Prince <span style="font-style: italic">coli</span> is added at t = 700, the number of the Prince <span style="font-style: italic">coli</span> does not increase greatly (Fig3). Therefore, C12 can exist until t = 70, and after that C12 decreases with an increase in AmiE (Fig4). |
| </p><br> | | </p><br> |
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| </p></div><br> | | </p></div><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 coli when t equals 700.<br><br> | + | <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> when t equals 700.<br><br> |
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| 4.5.2 ストーリーの再現<br> | | 4.5.2 ストーリーの再現<br> |
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| 5.2 Addition of other characters<br> | | 5.2 Addition of other characters<br> |
− | Initially, only 3 characters, Snow White, the Queen, and the Prince, appeared in our story. However, we got the simple question from junior high school and high school students that why other characters did not appear in our story, we decided to add others. We designed the Magic Mirror’s genetic circuit and conducted an experiment. Additionally, as dwarfs, we prepared the E. coli (strain name). As you can see in the photograph, they have round shapes compared to the general E. coli and are very charming. <br> | + | Initially, only 3 characters, Snow White, the Queen, and the Prince, appeared in our story. However, we got the simple question from junior high school and high school students that why other characters did not appear in our story, we decided to add others. We designed the Magic Mirror’s genetic circuit and conducted an experiment. Additionally, as dwarfs, we prepared the <span style="font-style: italic">E. coli</span> (strain name). As you can see in the photograph, they have round shapes compared to the general <span style="font-style: italic">E. coli</span> and are very charming. <br> |
| </p> | | </p> |
| <div align="center"><img src="url" height ="200"><br></div> | | <div align="center"><img src="url" height ="200"><br></div> |