Team:Tokyo Tech/Project

Project

1. Introduction

When you were very young, before going to sleep, have you ever asked your mother, “Mommy, read some picture books to me, please.”? You may have been read it aloud at least once.
Most stories for generations have lessons. The lessons from Snow White story are mainly the following two points.
Ⅰ It is not good to show off or to be jealous.
Ⅱ Do not trust unfamiliar people blindly.
It is not enough to be just friendly. A beautiful mind needs blessing with not only tenderness and richness but also a strong side that controls one’s feelings.
In our project, we will comprehensively evaluate Snow White story from the perspectives of two beauty, physical beauty and inner beauty.

2. Our goal is to evaluate Snow White Story in terms of real beauty.

“Magic Mirror on the wall, who is the fairest one of all?”

Once upon a time, there lived a Queen.
She was the fairest in the world and she herself also believed so.

Each time the Queen asked, “Magic Mirror on the wall, who is the fairest one of all?,” the mirror would give the same answer.
“You are the fairest one of all.”
This pleased the Queen greatly, for she knew that her Magic mirror could speak nothing but the truth and she asked it the same question.

“Magic Mirror on the wall, who is the fairest one of all?”

One winter night, the Queen asked her mirror as usual.
“Magic mirror on the wall, who is the fairest one of all?”
The mirror answered,
“Snow White is the fairest one.”

The Snow White was Queen’s daughter-in-law.
She was a kind and pure girl, and just turned seven years old.

Then the Queen was shocked, and beside herself with rage.
And thought,
“If I kill Snow White, I would be the fairest one of all again.”

The Queen prepared a poisoned apple.

She decided to transform into a Witch and give the apple to Snow White.
Snow White, who always takes people at their word , bit the apple, then sank into unconsciousness soon.

The Dwarfs found Snow White and they grieved her “death,” but they built a coffin and put her in it carefully.

One day, a Price from a neighboring country passed by the Dwarfs’ house.

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

This dislodged from Snow White’s throat the piece of poisoned apple that she had bitten off.


Awakened Snow White was adored by everyone and lived happily ever after.


THE END



The beauty is defined as follows in this project.

Beauty = physical beauty + inner beauty


In our project, we evaluate the real beauty of Snow White and the Queen with the fluorescence intensities of RFP and GFP.
We use the graph shown below to evaluate. The vertical axis of the graph shows the beauty of Snow White and the Queen, and the horizontal axis shows the elapsed time.
No matter how beautiful one’s physical appearance is, the one cannot be a real beauty unless has a beautiful mind. We will show you which woman has a real beauty, Snow White or the Queen.

Fig. 2-2-1.


Even if we intended to evaluate the real beauty, we cannot evaluate it unless we represent the story. At the very beginning, we represent the story.

3. Introduction of system forming the basis

Before introduction of our genetic circuit’s design, we will introduce a system that form the basis of our project.
They are TA system.
   What is TA system?
   The art of MazF / MazE
The expression of real beauty by MazE / MazF
MazE / MazF expression of real beauty
The creation of real beauty under magic MazE / MazF
The creation of real beauty enchanted by MazE / MazF
The creation of real beauty under the spell of MazE / MazF



3.1 What is TA system?

Toxin-antitoxin system


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. [1] Many prokaryotes have toxin-antitoxin systems, and organisms often have them in multiple copies. [2][3] 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.

一つ目のtoxin-anti-toxin : MazF / MazE


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. [4] One MazE dimer binds to two MazF dimers, thereby inactivates endoribonuclease activity of MazF dimer. [5] MazE is labile, subjected to degradation by ClpAP protease, whereas MazF is more stable. [6]

二つ目のtoxin-anti-toxin : YafO / YafN


YafO is a toxin protein, and YafN is its cognate antitoxin protein. [7] 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. [8] YafN is labile, subjected to degradation by Lon protease, whereas YafO is more stable [9].

The art of MazF / MazE

In our project, mazF and mazE are incorporated as a toxin and an antitoxin, respectively. Functioning 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, and 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.

4. Function

We conducted an experiment to confirm that the above four scenes can be represented. We introduced concepts in each scene and demonstrated them. Additionally, based on them, we simulated the representation of Snow White story.

   4.1. Scene1
   4.2. Scene2
   4.3. Scene3
   4.4. Scene4
   4.5. Modeling



4.1.　Scene1


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.

4.2. Scene2


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.



Fig. The intensity of Plux and Prhl promoters


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).



Fig.


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 which she has made by herself. Therefore, we made the mutants, using WT as a template.

4.3. Scene3


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.”



Fig.



Fig.


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.

4.4. Scene4


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.

4.5. Modeling

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.



Fig. 王子を初めから入れた場合の個体数



Fig. 王子を初めから入れた場合のAHL


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).



Fig. 王子をt = 700で投入した場合の個体数



Fig. 王子をt = 700で投入した場合のAHL


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.



Fig. time-dependent change of the concentrations of the two fluorescence proteins


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。

5．Integrated Human Practice

5.1 Snow White
5.2 Mirror
5.3 ACA


5.1 Snow White
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 to the public.

5.2 Addition of other characters
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, we decided to add others. 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 compared to the general E. coli and are very charming.


Fig. the Dwarf coli



Fig. ACA Dwarfs


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 the insertion of bases without considering.



Fig.


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.