“Magic mirror on the wall, who is the fairest one of all?”
The mirror answered,
“Snow White is the fairest one.”

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 Queen prepared a poisoned apple.
She decided to transform into a Witch and give the apple to Snow White.

The Queen, which has received C4HSL, produces LasI and MazF.
LasI produces signaling molecule, 3OC12HSL which represents the Poisoned Apple.
Additionally, produced MazF stops the translation in the Queen coli. MazE is incorporated into the genetic circuit so that MazE expression does not stop translation immediately. If the translation stops the moment MazF is expressed, sufficient amount of LasI will not be produced, and neither will 3OC12HSL. Then the Queen will fail the assassination of Snow White.

Snow White, who always takes people at their word, bit the apple, then sank into unconsciousness soon.

The basic design of Snow White coli’s genetic circuit is almost the same as the Queen coli’ s.
Snow White coli, which has received the C12HSL, the Poisoned Apple, expresses rhlI and MazF.
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.
In addition, expressed MazF stops the translation in the Snow White coli.

Although he knew her death, he couldn’t help lifting her up because of her beauty.
Then, she opened her eyes!

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.
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.

It is a cold inducible promoter (called Pcold commonly) that plays a most important role in this Scene. Unless the Magic Mirror answers the Queen’s question, this story never begins.

The experiment was conducted with BBa_1949001. We cultivated each sample at 18°C and 37°C and measured the GFP / Turbidity with a plate reader. The experimental result showed that samples cultured at 18°C had larger fluorescence intensity of GFP than those cultured at 37°C.
Thus, we found that the story begins by lowering the culture temperature.

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.

4.2.1 reporter assay
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.

Although C12 is added to Plux, we found that the promoter activity can be seen. In addition, the graph shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when added C4.

4.2.2 the simulation regarding the strength of rhl promoter
Using the experimental results of preceding paragraph, we simulated to confirm that the genetic circuit we designed works properly when using the existing rhl promoter.

The above diagram shows the intensity of the two promoters should be in the red region in the figure.
The above diagram shows the intensity of the two promoters should be in the red region in the figure. Because the green point shows the relationships of the promoters 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.
　　4.2.3　Rhl system Assay
We made mutants of rhl promoter by inserting a point mutation into the wild type (WT) rhl promoter.
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 (M) and Prhl the (NM).

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 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.

In this Scene, we introduce TA system as a concept.
If we do not show that translation is stopped by MazF, and translation is restarted by MazE, we cannot represent the key part of the story that "Snow White falls down by the Poisoned Apple and wake up again by the Prince.”

In this experiment, we used the BBa_K1949100 and Bba_1949102.
First, MazF was expressed by arabinose, and 2 h later, the MazE was expressed by IPTG.
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.
Moreover, when only MazF worksed, the RFU of GFP hardly rose, but when MazE was induced, the RFU of GFP rose. (link: toxin assay)
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.
According to the experiments, we showed that TA system works properly.

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 3OC12HSL by the Prince coli. Therefore, we examined whether AmiE selectively degrades AHLs.

Fig. AmiE degrades C12   Fig. AmiE barely degrades C４
C12 is greatly degraded. On the other hand, the C4 is barely degradedPrhl(LR).

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.

From the results of Wet , 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.

4.5.1　王子様いつやってくのだろうか
We simulated to confirm which is better, adding the Prince coli first or adding it in midstream, 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 degraded too much. Then, C12 cannot exist in the medium (Fig. 2) and the circuit does not work correctly.

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).

From this result, it was found that the genetic circuit works well by adding the Prince coli when t equals 700.

　　 4.5.2　ストーリーの再現
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.

シミュレーションをもとに、白雪姫と女王様のどちらが真に美しいのか見てみましょう。

In the blue area of Fig, ここでは、白雪姫の蛍光強度が、女王様の蛍光強度を上回っています。
ある日を境に、白雪姫の美しさが女王の美しさを超えたからです。It represents Snow White got fairer more and more.
In the pink area of Fig, 投入されたC4によりC12が生産さます。これによりトキシンであるMazFが継母の個体内で増加し、蛍光タンパク質の増加を抑制します。It looks as if after mirror’s answer Queen transformed into a Witch and gave the apple to Snow White.これは女王様が嫉妬に怒り狂い「犯罪」に手を染めてしまった場面にあたります。
In the green area of Fig, C12が増加してC12によって誘導される白雪姫内のMazFがより増加し、GFPがRFPを上回ります。It looks as if Snow White bit the apple, then sank into unconsciousness soon
In the yellow area of Fig, 投入された王子が生産したAmiEによりC12が分解されるため白雪姫のMazFが減少してC4が増加します。It looks as if Prince lifted Snow White and she opened her eyes.
以上より、私たちがデザインした回路はストーリーを再現でき、また、真に美しいのは白雪姫であるということがわかりました。