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| Snow White, who always takes people at their word, bit the apple, then sank into unconsciousness soon.<br> | | Snow White, who always takes people at their word, bit the apple, then sank into unconsciousness soon.<br> |
| -----<br><br> | | -----<br><br> |
− | The basic design of Snow White coli ’s genetic circuit is almost the same as the Queen coli’s. | + | The basic design of Snow White coli ’s genetic circuit is almost the same as the Queen <span style="font-style : italic">coli</span>’s. |
− | Snow White coli, which has received the C12HSL the Poisoned Apple, expresses hlI and MazF. | + | Snow White <span style="font-style : italic">coli</span>, which has received the C12HSL the Poisoned Apple, expresses hlI and MazF. |
− | Produced RhlI synthesizes signaling molecule C4HSL. As was described in the introduction of the Queen in the previous section, the Queen coli can receive C4HSL and monitor Snow White coli through this molecule. | + | Produced RhlI synthesizes signaling molecule C4HSL. As was described in the introduction of the Queen in the previous section, the Queen <span style="font-style : italic">coli</span> can receive C4HSL and monitor Snow White <span style="font-style : italic">coli</span> through this molecule. |
− | In addition, MazF expression inhibits the translation in Snow White coli.<br> | + | In addition, MazF expression inhibits the translation in Snow White <span style="font-style : italic">coli</span>.<br> |
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| Then, she opened her eyes!<br> | | Then, she opened her eyes!<br> |
| -----<br><br> | | -----<br><br> |
− | The Prince coli which has received 3OC12HSL expresses AmiE. In general, it is said that AmiE degrades HSL with more than 7 carbons. This leads to the degradation of the Poisoned Apple 3OC12HSL. | + | The Prince <span style="font-style : italic">coli</span> which has received 3OC12HSL expresses AmiE. In general, it is said that AmiE degrades HSL with more than 7 carbons. This leads to the degradation of the Poisoned Apple 3OC12HSL. |
− | When the Poisoned Apple is degraded, MazF expression is inhibited in Snow White, and gradually its function is counteracted by MazE. Then, translation restarts in Snow White coli. | + | When the Poisoned Apple is degraded, MazF expression is inhibited in Snow White, and gradually its function is counteracted by MazE. Then, translation restarts in Snow White <span style="font-style : italic">coli</span>. |
| <br> | | <br> |
| </p><br> | | </p><br> |
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| <p class="normal_text">5.2 The screening of rhl promoter with optimal strength for Scene 2<br> | | <p class="normal_text">5.2 The screening of rhl promoter with optimal strength for Scene 2<br> |
− | The Queen coli gave the Poisoned Apple, and Snow White coli falls asleep. We use cell-cell communication to represent this scene, so we need the reporter with optimal strength to make the genetic circuit designed by us work properly. | + | The Queen <span style="font-style : italic">coli</span> gave the Poisoned Apple, and Snow White <span style="font-style : italic">coli</span> falls asleep. We use cell-cell communication to represent this scene, so we need the reporter with optimal strength to make the genetic circuit designed by us work properly. |
| Therefore, 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 obtaining of optimal rhl promoter mutants for our project. <br> | | Therefore, 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 obtaining of optimal rhl promoter mutants for our project. <br> |
| </p> | | </p> |
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| In the experiment, we added reagent AHLs into the reporters and examined their fluorescence intensities. | | In the experiment, we added reagent AHLs into the reporters and examined their fluorescence intensities. |
| As a result, we succeeded in newly obtaining stronger mutants than the WT Prhl (NM).<br> | | As a result, we succeeded in newly obtaining stronger mutants than the WT Prhl (NM).<br> |
− | iGEM Tokyo_Tech has Improved rhl promoter in the past. However, comparing the Prhl (NM) to Prhl (LR), the SN ratio of Prhl (NM) was higher than that of Prhl (LR). Futuremore, the result shows that Prhl (LR)has crosstalk with C12-AHL at a constant rate. When representing Snow White story, and the crosstalk to C12 happens, the Queen coli will suicide by eating the Poisoned Apple made by herself. Therefore, we made the mutants using WT as a template.<br> | + | iGEM Tokyo_Tech has Improved rhl promoter in the past. However, comparing the Prhl (NM) to Prhl (LR), the SN ratio of Prhl (NM) was higher than that of Prhl (LR). Futuremore, the result shows that Prhl (LR)has crosstalk with C12-AHL at a constant rate. When representing Snow White story, and the crosstalk to C12 happens, the Queen <span style="font-style : italic">coli</span> will suicide by eating the Poisoned Apple made by herself. Therefore, we made the mutants using WT as a template.<br> |
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| <p class="normal_text">5.4 AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene 4<br> | | <p class="normal_text">5.4 AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene 4<br> |
| In the final Scene, we introduce the selective degradation of AHLs by AmiE as a concept. | | In the final Scene, we introduce the selective degradation of AHLs by AmiE as a concept. |
− | As shown in the TA system, we found that Snow White can wake up again because MazE counteracts the function of MazF. However, we have not showed the trigger, that is, the degradation of only 3OC12HSL by the Prince coli. Therefore, we examined whether AmiE selectively degrades AHLs.<br> | + | As shown in the TA system, we found that Snow White can wake up again because MazE counteracts the function of MazF. However, we have not showed the trigger, that is, the degradation of only 3OC12HSL by the Prince <span style="font-style : italic">coli</span>. Therefore, we examined whether AmiE selectively degrades AHLs.<br> |
| 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. | | 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. |
| For these reasons, we showed that AmiE selectively degrades AHLs, only C12 in this project.<br> | | For these reasons, we showed that AmiE selectively degrades AHLs, only C12 in this project.<br> |
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| <p class="normal_text">5.5.1. When the Prince comes?<br> | | <p class="normal_text">5.5.1. When the Prince comes?<br> |
− | We simulated to confirm which is better, the Prince coli exist from the beginning, that is, the Prince has known Snow White and watches her grow or he comes across her, in order to make our genetic circuit work. | + | We simulated to confirm which is better, the Prince <span style="font-style : italic">coli</span> exist from the beginning, that is, the Prince has known Snow White and watches her grow or he comes across her, in order to make our genetic circuit work. |
− | 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 overdegraded. Then, C12 cannot exist in the medium (Fig. 2), and the circuit does not work correctly.<br> | + | 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 overdegraded. Then, C12 cannot exist in the medium (Fig. 2), and the circuit does not work correctly.<br> |
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− | 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).<br> | + | 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).<br> |
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− | From this result, it was found that the genetic circuit works well by adding the Prince coli at t=700. In other words, as with the original story, the Prince coli comes across Snow White coli at t=700 and rescues her.<br> | + | 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> |
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| </p><br> | | </p><br> |
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| 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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− | Based on the simulation, we will show you which one is a real beauty, Snow White coli or the Queen coli.<br> | + | Based on the simulation, we will show you which one is a real beauty, Snow White <span style="font-style : italic">coli</span> or the Queen <span style="font-style : italic">coli</span>.<br> |
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− | In the blue area of Fig, the fluorescence intensity of Snow White coli exceeds the Queen coli’s. | + | In the blue area of Fig, the fluorescence intensity of Snow White <span style="font-style : italic">coli</span> exceeds the Queen <span style="font-style : italic">coli</span>’s. |
− | That is because one day, Snow White coli’s beauty exceeded the Queen coli’s beauty. Snow White got fairer and fairer.<br> | + | That is because one day, Snow White <span style="font-style : italic">coli</span>’s beauty exceeded the Queen <span style="font-style : italic">coli</span>’s beauty. Snow White got fairer and fairer.<br> |
− | In the pink area of Fig, added C4 leads to production of C12. Because of this, MazF, a toxin, increases in the Queen coli, and suppresses increase of fluorescence protein. It looks as if after the Magic Mirror’s answer, the Queen transformed herself into a Witch and gave the Poisoned Apple to Snow White. This is the scene where the Queen is mad with jealousy and drifts into crime<br> | + | In the pink area of Fig, added C4 leads to production of C12. Because of this, MazF, a toxin, increases in the Queen <span style="font-style : italic">coli</span>, and suppresses increase of fluorescence protein. It looks as if after the Magic Mirror’s answer, the Queen transformed herself into a Witch and gave the Poisoned Apple to Snow White. This is the scene where the Queen is mad with jealousy and drifts into crime<br> |
− | In the green area of Fig, C12 increases, and MazF induced by C12 increases in Snow White coli, which causes GFP excess over RFP. It looks as if Snow White bit the apple, then sank into unconsciousness soon<br> | + | In the green area of Fig, C12 increases, and MazF induced by C12 increases in Snow White <span style="font-style : italic">coli</span>, which causes GFP excess over RFP. It looks as if Snow White bit the apple, then sank into unconsciousness soon<br> |
− | In the yellow area of Fig, since AmiE produced by the added Prince coli degrades C12, MazF in Snow White coli decreases and increases C4. It looks as if the Prince lifted Snow White and she opened her eyes.<br> | + | In the yellow area of Fig, since AmiE produced by the added Prince <span style="font-style : italic">coli</span> degrades C12, MazF in Snow White <span style="font-style : italic">coli</span> decreases and increases C4. It looks as if the Prince lifted Snow White and she opened her eyes.<br> |
− | From the above, we were able to represent the story by the genetic circuit we designed, and furthermore, it was found that Snow White coli is a real beauty. <br> | + | From the above, we were able to represent the story by the genetic circuit we designed, and furthermore, it was found that Snow White <span style="font-style : italic">coli</span> is a real beauty. <br> |
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