Project

1. Introduction

When you were little, before going to sleep, you have probably asked your mother, "Mommy, read some picture books to me, please?" Your mother may have read aloud many stories. Most of the stories that have been told for generation contain lessons in them. The lessons from the Snow White story are mainly the following two points.

Ⅰ. Do not show off or envy someone.

Ⅱ. Do not trust unfamiliar people blindly.

For a beautiful mind, we need not only to be kind and generous but also to keep a strong heart and control ourselves.In our project, the Snow White story is going to be recreated and Snow White and the Queen is going to be evaluated in terms of the beauty from both inside and outside.

2. Our goal is to evaluate the real beauty of Snow White and the Queen

"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!

because this action 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

In our project, we evaluate the real beauty of Snow White and the Queen with the fluorescence intensities of RFP (Red Fluorescence Protein) and GFP (Green Fluorescence Protein). The graph shown below is used for evaluation; the vertical axis shows the beauty of Snow White and the Queen, and the horizontal axis shows the elapsed time.

The Real beauty is defined as the followings in this project.

No matter how beautiful the appearance is, no one can be a real beauty without a beautiful mind. We will show you which woman has the real beauty, Snow White or the Queen.

Real Beauty = outer beauty + inner beauty

To evaluate the Real beauty, we are going to recreate the Snow White story using E. coli.

Fig. 2-2-1. Time-dependent change of the concentrations of fluorescent protains

To evaluate the real beauty, we recreated Snow White story by using E.coli.

3. Introduction of system forming the basis

We will first introduce the system that forms the basis of our project, the 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. Many prokaryotes harbor toxin-antitoxin systems on the genomes, typically in multiple copies. Changes in the physiological conditions, such as stress conditions or viral infection trigger antitoxin degradation by cytosolic proteases. 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 bacteria to survive in unfavorable conditions. In general, toxin proteins are more stable than antitoxin proteins, but antitoxins are expressed at a higher level in cells.

First toxin‐antitoxin: MazF / MazE

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

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

Second toxin‐antitoxin: YafO / YafN

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 acquires endoribonuclease activity. YafO and YafN form a complex, resulting in neutralization of YafO. YafN is labile and is subjected to degradation by the Lon protease, whereas YafO is more stable.

3.2 The art of MazF / MazE

In our project, MazF and MazE are incorporated in a single cell 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 fluorescence.

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

Fig. 2-3-2-2

Fig. 2-3-2-3

Fig. 2-3-2-1

When MazF expression is induced, the mRNA of GFP is cleaved, and thus 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. Our genetic circuit design

Next, we will introduce the genetic circuits that we have designed.

Our project begins from the scene where the Magic Mirror answers the Queen's question.

We will split the story into 4 scenes and introduce how our genetic circuits work in each scene.

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 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.

4.2 Scene2 : The Queen’s trap

------
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

-----

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.

4.4. Scene4 : The Prince’s rescue

-----
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.

5. Replication of “Snow White” by our genetic circuit

We conducted an experiment and simulated to confirm that the above four scenes can be represented. 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(cslled Pcold commonly) that plays a most important role in this Scene. Yhis story never begins unless the Magic Mirror answers the Queen's question.

The experiment was conducted by BBa_1949001(cold-inducible promoter). 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 18°C had higher fluorescence intensity of GFP than those cultured at 37°C.

Thus, it has been confirmed that the story could be begun with lowering the culture temperatures

Read Pcold Assay page .

5.2 The screening of Prhl with optimal strength for Scene 2

Eating the Poisoned Apple given by the Queen coli, the Snow White coli falls asleep. We exploit the cell-cell communication mechanism of bacteria to represent this scene, and so we need promoters with optimal strength. Otherwise, our final genetic circuit never 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, we evaluated the activities the existing promoters, Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) by observing the reactivity to three AHLs (= expression inducers, C4HSL, 3OC6HSL, and 3OC12HSL).

However, 3OC12HSL reacted to Prhl and Plux promoters, the latter of which is not the native combination of inducer-promoter, and this phenomenon is called “crosstalk”. The graph also shows that Prhl has a large leak, and the Prhl activity is almost independent on C4HSL addition.

Read AHL Reporter Assay.

5.2.2 Simulation regarding Prhl strength　

Based on the experimental results in the preceding paragraph, we performed a simulation to analyze whether our genetic circuit works properly when the existing Prhl was used.

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.

Read Model page.

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.

Read Rhl System Assay .

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.

Read mazEF System Assay.

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.

Read AmiE Assay.

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.

Read Model page.

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.

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 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.

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