A toxin-antitoxin system is composed of two or more cognate genes that encode toxins and antitoxins. Toxins are proteins, whereas antitoxins are either proteins or non-coding RNAs. Many prokaryotes have toxin-antitoxin systems, and organisms often have them in multiple copies. Changes in the physiological conditions, such as stress conditions or viral infection leads to the antitoxin degradation, and proteases degrade many antitoxins. Unleashed toxin proteins impede or alter cellular processes including translation, cell division, DNA replication, ATP synthesis, mRNA stability or cell wall synthesis and lead to dormancy. This dormant state probably enables the bacteria to survive in unfavorable conditions. In general, toxin is more stable than antitoxin, but antitoxin is expressed to a higher level.

MazF is a toxin protein, and MazE is its cognate antitoxin protein. MazF is a ribosome-independent endoribonuclease. This endoribonuclease activity leads to bacterial growth arrest. MazE and MazF form homodimers, and MazF dimer cleaves mRNAs at ACA sites. One MazE dimer binds to two MazF dimers, thereby inactivates endoribonuclease activity of MazF dimer. MazE is labile, subjected to degradation by ClpAP protease, whereas MazF is more stable.

YafO is a toxin protein, and YafN is its cognate antitoxin protein. YafO is a ribosome-associated mRNA interferase that cleaves mRNAs’ downstream 11‐13 bases of the translation initiation sites. When 70S ribosome is dissociated, YafO associates with 50S ribosome subunit and gets endoribonuclease activity. YafO and YafN forms a complex Via binding with YafN, YafO is neutralized. YafN is labile, subjected to degradation by Lon protease, whereas YafO is more stable.

In our project, mazF and mazE are incorporated as a toxin and an antitoxin, respectively. Functions of these two proteins makes it possible to evaluate the beauty of Snow White with GFP and RFP.

We will give you an example for a better understanding. Imagine that RFP is expressed little by little in E. coli.

When MazF expression is induced, the mRNA of GFP is cut, so GFP cannot be translated. Additionally, when MazE is induced, MazE and MazF forms a complex. MazF loses its function, which restarts GFP translation.

In this way, we can control protein translation. That's why we decided to represent Snow White story.

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The story starts with the scene where it snows and gets cold. The Magic Mirror coli can produce RhlI protein under low temperature condition. RhlI protein leads to the production of a signaling molecule C4HSL, which is received by the Queen coli telling that Snow White is the fairest of the all.

The Queen, which has received C4HSL, produces LasI and MazF.

LasI produces a signaling molecule, 3OC12HSL which is the Poisoned Apple.

Additionally, produced MazF inhibits the translation in the Queen. If the translation is inhibited 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.

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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'. Snow White coli receives 3OC12HSL, the Poisoned Apple, and expresses RhlI and MazF. RhlI synthesizes signaling molecule C4HSL. As described in the previous section, the Queen coli receives C4HSL and monitor Snow White coli through it. MazF, on the other hand, inhibits the translation in Snow White coli.

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

The Prince coli which receives 3OC12HSL expresses AmiE. AmiE is said to degrade HSL with more than 6 carbons, which means that AmiE degrades the Poisoned Apple, 3OC12HSL.By the degradation of the Poisoned Apple, MazF expression is inhibited in Snow White and its function is counteracted by MazE. Then, translation restarts in Snow White coli.

In this scene, the cold inducible promoter, so called Pcold/cold promoter, plays an important role in this Scene. This story never begins unless the Magic Mirror answers the Queen’s question.

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 37°C.

Thus, we found that the story begins by lowering the culture temperature.

5.2 The screening of Prhl with optimal strength for Scene 2

Eating the Poisoned Apple given by the Queen coli, 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.

Therefore, we introduce rhl system assay as a concept in this Scene. We improved the function of Prhl through the process listed below and succeeded in newly obtaining of optimal Prhl mutants for our project.

5.2.1　Reporter assay

First of all, we evaluated the activities of 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.

5.2.2 Simulation regarding Prhl strength　

Using the experimental results of preceding paragraph, we simulated to confirm that the genetic circuit which we designed works properly when using the existing Prhl.

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.

5.2.3　Improvement of Prhl
We made mutants of Prhl by inserting a point mutation into the wild type (WT) Prhl. 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).
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 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 inhibited 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/p.

From the experimental results, we found that the turbidity of samples without MazE 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 found that MazF stops cell growth and translation of E. coli, but MazE restarts cell growth that have stopped and furthermore restarts translation.

5.4 AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene 4
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.
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.

5.5　the simulation related to the story
From the results of wet lab, 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.

5.5.1. When the Prince comes?
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. 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.
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 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.

5.5.2　representation of the story
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.
Based on the simulation, we will show you which one is a real beauty, Snow White coli or the Queen coli.
In the blue area of Fig, the fluorescence intensity of Snow White coli exceeds the Queen coli’s. That is because one day, Snow White coli’s beauty exceeded the Queen coli’s beauty. Snow White got fairer and fairer.
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
In the green area of Fig, incereased C12 induces MazF in Snow White coli, which causes GFP excess over RFP. It looks as if Snow White bit the apple, then sank into unconsciousness soon
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.
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.

Why we are going to participate in the iGEM with the theme of Snow White? It is resulted from drawing on the opinion obtained through various dialogues with the public. This time, we decided to deal with Snow White, which was easy-to-follow, familiar, and attracted the public.

6.2 Addition of other characters

6.3. The software development for future work