Here we indicate that the cold inducible promoter is a promoter that depends mainly on temperature.
a Pcold-gfp was constracted.

In this experiment, each sample was cultivated at 18°C and 37°C, after that their fluorescence intensity was measured.
From the experiment results it was found that the fluorescence intensity of the gfp of the samples cultivated at 18°C was higher than the ones cultivated at 37°C.
From the above result, we are able to show that this promoter is a promoter induced by low temperatures.

Here we indicate how we produced the rhl promoter that suited our project best

A point mutation was inserted in a wild type rhl promoter.
In the experiment, an AHL reagent was included in the reporter, and then the fluorescence intensity was measured.
As a result, the mutant promoters Prhl(M) and Prhl(NM), which is stronger than a wild type promoter, were newly obtained.

iGEM Tokyo_Tech has improved the rhl promoter in previous years. However, comparing the Prhl(NM) to the Prhl(LR), Prhl(NM) did not show any crosstalk with C12 and the SN ratio of Prhl(NM) was stronger than that of Prhl(LR). Also, the experiment showed that Prhl(LR) crosstalks with C12-AHL at a specific proportion. If we use it in the recreation of Snow White,and if the promoter has crosstalks with C12, it means that the Queen eats the poisoned apple, she herself produced, and dies. So this is not a promoter we can use in our project.
The graph also shows that Prhl(NM) almost doesn´t have crosstalk with C12.
Base on the above results, we can say that we found a promoter with high intensity, and it doesn´t have crosstalk with C12, which is the most suitable for our story.

Here we indicate the inhibition of the E. Coli´s multiplication and inhibition of translation by MazF and the restarting of them by MazE.
We constructed a circuit that includes mazEF .

In this experiment, MazF was expressed by arabinose , and after 2 hours MazE was expressed by IPTG.
From the experiment results, it was found that the turbility of the sample without MazE almost didn’t rise at all. However, it was also found that if IPTG is added, and the n MazE is expressed, the E. Coli will restart its multiplication.
Also, if only the MazF is working, the RFU of GFP barely rise But if MazE is induced, the RFU of GFP will rise. (Link: Toxin assay)
As the result above, it was concluded that MazF interrupts the multiplication of E. Coli and translation. However, MazE restarted the multiplication and translation stopped by MazF.
Through this experiment, we showed that the TA system functions properly.

iGEM Tokyo_Tech has improved the rhl promoter in previous years. However, comparing the Prhl(NM) to the Prhl(LR), Prhl(NM) did not show any crosstalk with C12 and the SN ratio of Prhl(NM) was stronger than that of Prhl(LR). Also, the experiment showed that Prhl(LR) crosstalks with C12-AHL at a specific proportion. If we use it in the recreation of Snow White,and if the promoter has crosstalks with C12, it means that the Queen eats the poisoned apple, she herself produced, and dies. So this is not a promoter we can use in our project.
The graph also shows that Prhl(NM) almost doesn´t have crosstalk with C12.
Base on the above results, we can say that we found a promoter with high intensity, and it doesn´t have crosstalk with C12, which is the most suitable for our story.

Here we indicate that the AmiE decomposes AHL selectively.

In this experiment, it was examined whether AHL would be decomposed after the insertion of 3OC12HSL and C4HSL molecules into a culture of E. Coli that express AmiE.
From the experiment results, it was found that the C12 inserted into a culture of AmiE expressing E. Coli is greatly decomposed. On the other hand, the C4 inserted in this culture, is barely decomposed.
From the above result, we were able to show that AmiE selectively decomposes AHL molecules.