First toxin‐antitoxin: MazF / MazE

Fig. 2-3-1-1. TA system

Second toxin‐antitoxin: YafO / YafN

Fig. 2-3-2-3

Fig. 2-3-2-1

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

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

4.3 Scene3 : Snow White's sleep

4.4. Scene4 : The Prince’s rescue

5.2.3　Improvement of Prhl
We generated mutants of Prhl by introducing a point mutation into the wild type (WT) Prhl. In the experiment, we added reagent AHLs into the reporter cells and examined their fluorescence intensities. As a result, we succeeded in newly obtaining stronger mutants than the WT Prhl. The new mutant was named Prhl (NM).

Actually, iGEM Tokyo_Tech has improved Prhl in the past, yielding Prhl (LR). When comparing the Prhl (NM) to Prhl (LR), the SN ratio of Prhl (NM) was higher than that of Prhl (LR), and even better, Prhl (NM) showed lower crosstalk with 3OC12HSL. When representing the Snow White story, in the presence of the crosstalk to 3OC12HSL, the Queen coli have to suicide by eating the Poisoned Apple made by herself. Therefore, this is the other reason why we generated 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.

According to the experiments, we showed that TA system works properly.

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.

As a result, if we introduce the Prince coli from the beginning, the number of Prince coli increases too much (Fig.5-4-1-1) so the AmiE the Prince coli produces augments and the decomposition of C12 also occurs overly. So C12 is almost inexistent in the medium (Fig.5-4-1-2).

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.5-2-2, the concentration of fluorescent proteins start to increase. The concentration of RFP of Snow White coli exceeds that of GFP of the Queen coli.
It is as if Snow White got fairer more and more.

In the pink area of Fig.5-2-2, the concentration of C12 increase thanks to the appearance of C4. As a result, the MazF inside Snow White coli and the Queen coli start to suppress the increment of fluorescet proteins.
It is as if the Queen, influenced by the Mirror's answer, transforming into a Witch in order to give Snow White a poisoned apple.

In the green area of Fig.5-2-2, the concentration of C12 more increases and the MazF inside Snow White coli more suppress the increment of GFP. So the concentration GFP exceeds that of RFP.
It looks as if Snow White bit the apple, sinking into unconsciousness soon.

In the yellow area of Fig.5-2-2, the AmiE synthesized by the introduced Prince coli decomposes C12 so the MazF inside Snow White coli diminishes and the concentration of GFP resumes.
It looks as if the Prince lifted Snow White and she opened her eyes.

Initially, only 3 characters, Snow White, the Queen, and the Prince, appeared in our story. However, we got the simple question from junior high school and high school students that why other characters did not appear in our story. Then we decided to add other characters. We designed the Magic Mirror’s genetic circuit and conducted an experiment. Additionally, as dwarfs, we prepared the E. coli (strain name). As you can see in the photograph, they have round shapes and are very charming compared to the general E. coli.

One students asked the question, "Can you apply this project to contribute society?" After we thought what we could do, we decided to have a dialogue with an expert.

We developed a new software named ACA Dwarfs. This software helps to control the sensitivity of the protein to MazF by regulating the number of ACA sequences in the mRNA sequence. ACA Dwarfs can increase or decrease the number of ACA sequences on mRNA without changing amino acid sequences that the mRNA specifies or frameshift resulted from insertion of bases without considering.

6.3.2 ACA Dwarfs

After having dialogue with experts, we obtained a comment that TA system has a potential to be linked to development of effective technology. However, at present, there exits problems when using a TA system to control the protein production.

We cannot selectively produce only desired protein because other proteins would be also produced at the same time. Then, our dry lab used Java to develop software named “ACA Dwarfs” adjusting number of ACA base sequence as a solution to the problem.

This graph shows that the simulation result when changing the base sequences of GFP and RFP with ACA Dwarfs. In case of GFP and RFP, their concentrations decreases earlier than original ones.
Using ACADwarfs enabled us to make GFP and RFP more reactive to MazF.