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| <p class="normal_text">The experiment was conducted by BBa_1949001. We cultivated each sample at 18°C or 37°C and measured the [GFP / Turbidity] with a plate reader. The experimental result showed that samples cultured at 18°C had higher fluorescence intensity of GFP than those cultured at 37°C.</p> | | <p class="normal_text">The experiment was conducted by BBa_1949001. We cultivated each sample at 18°C or 37°C and measured the [GFP / Turbidity] with a plate reader. The experimental result showed that samples cultured at 18°C had higher fluorescence intensity of GFP than those cultured at 37°C.</p> |
| <p class="normal_text">Thus, we found that the story begins with lowering the culture temperature .</p> | | <p class="normal_text">Thus, we found that the story begins with lowering the culture temperature .</p> |
| + | <a href="https://2016.igem.org/Team:Tokyo_Tech/Promoter_Assay/Pcold ">Read Pcold Assay page </a>. |
| <br><br></p> | | <br><br></p> |
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| <div align="center"><img src="https://static.igem.org/mediawiki/2016/0/00/T--Tokyo_Tech--assay_ahl_reporter.png" height ="200"></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/0/00/T--Tokyo_Tech--assay_ahl_reporter.png" height ="200"></div> |
| <p class="normal_text">Even adding 3OC12HSL to Plux promoter, the promoter activity was still observed. The graph also shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when C4HSL is added.</p> | | <p class="normal_text">Even adding 3OC12HSL to Plux promoter, the promoter activity was still observed. The graph also shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when C4HSL is added.</p> |
| + | <a href="https://2016.igem.org/Team:Tokyo_Tech/AHL_Assay/AHL_Reporter_Assay |
| + | ">Read AHL Reporter Assay</a>. |
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| <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> | | <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> |
| + | <a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prhl ">Read Model page</a>. |
| </p><br> | | </p><br> |
| </p><br> | | </p><br> |
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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 Prhl 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> | + | iGEM Tokyo_Tech has improved Prhl 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.<a href="https://2016.igem.org/Team:Tokyo_Tech/AHL_Assay/Rhl_System_Assay ">Read Rhl System Assay </a>.<br> |
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| 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> |
| </p><p class="normal_text">From the above, we found 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.</p> | | </p><p class="normal_text">From the above, we found 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.</p> |
− | According to the experiments, we showed that TA system works properly.</p> </p><br> | + | 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>. |
| + | </p><br> |
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| 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> | | 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 <span style="font-style : italic">E. coli</span> 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 <span style="font-style : italic">E. coli</span> 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><a href="https://2016.igem.org/Team:Tokyo_Tech/AmiE_Assay |
| + | ">Read AmiE Assay</a>. |
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| 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> | | 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 <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> | + | 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><a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prince_coli ">Read Model page</a>. |
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| </p><br> | | </p><br> |
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| In the green area of Fig, incereased C12 induces MazF 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 green area of Fig, incereased C12 induces MazF 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 <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> | | 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 <span style="font-style : italic">coli</span> 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><a href="https://2016.igem.org/Team:Tokyo_Tech/Model#results">Read Model page</a>. |
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