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 envy someone.
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. Story

“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.

3.1 What is TA 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.

First toxin‐antitoxin: 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]

Second toxin‐antitoxin: 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. 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.

4．Explanation of the circuit designed by us

We introduce the genetic circuits which we designed. Our project begins from the scene where the Magic Mirror answers the Queen’s question. We pick up 4 famous scenes including this scene and will introduce genetic circuits in each scene in order.

4.1 Scene1 : The magic mirror’s answer

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

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 production leads to the production of the signaling molecule, C4HSL which tells the Queen that Snow White is the fairest. the Queen coli receives this molecule.

4.2 Scene2 : The Queen’s trap

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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 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.

4.3 Scene3 : Snow White's sleep

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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’s. Snow White coli, which has received the C12HSL the Poisoned Apple, expresses RhlI and MazF. Produced RhlI synthesizes signaling molecule C4HSL. As was described in the introduction of the Queen in the previous section, the Queen coli can receive C4HSL and monitor Snow White coli through this molecule. In addition, MazF expression inhibits the translation in Snow White coli.

4.4. Scene4 : The Prince’s rescue

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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 has received 3OC12HSL expresses AmiE. In general, AmiE degrades HSL with more than 7 carbons. This leads to the degradation of the Poisoned Apple 3OC12HSL. When the Poisoned Apple is degraded, MazF expression is inhibited in Snow White, and gradually MazF function is counteracted by MazE. Then, translation restarts in Snow White coli.

5. Q.E.D. for the representation of Snow White

We conducted an experiment and simulated 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.

5.1 Cold inducible promoter functions at 18°C in Scene 1
It is a cold inducible promoter (called Pcold commonly) that plays a most important role in this Scene. This story never begins unless the Magic Mirror answers the Queen’s question.

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 higher fluorescence intensity of GFP than those cultured at 37°C. Thus, we found that the story begins by lowering the culture temperature.

5.2 The screening of rhl promoter 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 rhl promoter through the process listed below and succeeded in newly obtaining of optimal rhl promoter 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.

The Queen coli, 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.

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 rhl promoter.

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 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 (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-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.

5.3 MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3

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.
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.

6．Integrated Human Practice

In promoting our project, we had dialogues with the public and experts. Based on the opinions from them, we developed our project. This led to success in creation of a well-rounded project with the connection between the public and experts, keeping from our narrow view.

6.1 “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 the public.

6.2 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. 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.

6.3. The software development for future work

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.

6.3.1 The 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 適当に塩基を挿入した結果の

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.

7. Reference

[1] Unterholzner SJ, Poppenberger B, Rozhon W. Toxin-antitoxin systems: Biology, identification, and ampicillin. Mob Genet Elements. 2013 Sep; 3(5): e26219.

[2]Zielenkiewicz U., Ceglowski P. The toxin-antitoxin system of the streptococcal plasmid pSM19035.J. Bacteriol. 2005;187:6094–6105.

[3] Fozo EM, Makarova KS, Shabalina SA, Yutin N, Koonin EV, Storz G. Abundance of type I toxin–antitoxin systems in bacteria: searches for new candidates and discovery of novel families. Nucleic Acids Res. 2010 Jun; 38(11): 3743–59.

[4] Gerdes K, Wagner EG. RNA antitoxins. Curr. Opin. Microbiol. 2007 Apr; 10 (2): 117–24.

[5] Zhang J., Y Zhang, L Zhu, Suzuki M, Inouye M. Interference of mRNA function by sequence-specific endoribonuclease PemK. J. Biol. Chem. 2004 Mar; 279:20678-20684.

[6] Park J.-H., Yamaguchi Y., Inouye M. Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases. Appl. Environ. Microbiol. 2012 Jun; 78(11): 3794–3799.

[7] Aizenman E., H Engelberg-Kulka, G Glaser. An Escherichia coli chromosomal “addiction module” regulated by guanosine 3′,5′-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci U S A. 1996 Jun; 93(12): 6059–6063.

[8] Brown J. M., and Shaw K. J. A Novel Family of Escherichia coli Toxin-Antitoxin Gene Pairs. J Bacteriol. 2003 Nov; 185(22): 6600–6608.

[9] Zhang Y, Yamaguchi Y, Inouye M. Characterization of YafO, an Escherichia coli toxin. J Biol Chem. 2009 Sep; 284(38): 25522–25531.

[10]Christensen-Dalsgaard M., Jorgensen M.G., Gerdes K. Three new RelE-homologous mRNA interferases of Escherichia coli differentially induced by environmental stresses. Mol. Microbiol. 2010 Jan; 75:333–348.

[11]Ochiai S, Yasumoto S, Morohoshi T, Ikeda T. AmiE, a Novel N-Acylhomoserine Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24. Appl Environ Microbiol.2014 Nov;80(22):6919-25.

[12] Gerardo Medina et al. Mechanism of Pseudomonas aeruginosa RhlR Transcriptional Regulation of the rhlAB Promoter. J Bacteriol. 2003 Oct; 185(20): 5976–5983.

Team:Tokyo Tech/Project