Latest revision as of 05:24, 27 November 2016

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.

@@ Line 99: / Line 99: @@
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 				<h3 class="link"><a href="#sce4"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;4.4. Scene4 : The Prince's rescue</span></a></h3>
 				<h3 class="link"><a href="#Q.E.D.">5. Q.E.D. for the representation of Snow White</a></h3>
-				<h3 class="link"><a href="#5-sce1"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.1. Cold inducible promoter functions at 18°C in Scene 1</span></a></h3>
+				<h3 class="link"><a href="#sysscene1"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.1. Cold inducible promoter functions at 18°C in Scene 1</span></a></h3>
-				<h3 class="link"><a href="#5-sce2"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.2. The screening of rhl promoter with optimal strength for Scene 2</span></a></h3>
+				<h3 class="link"><a href="#sysscene2"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.2. The screening of rhl promoter with optimal strength for Scene 2</span></a></h3>
 				<h3 class="link"><a href="#ass"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5.2.1. reporter assay</span></a></h3>
 				<h3 class="link"><a href="#sml"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5.2.2. Simulation regarding Prhl strength</span></a></h3>
 				<h3 class="link"><a href="#imp"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5.2.3. Improvement of Prhl</span></a></h3>
-				<h3 class="link"><a href="#5-sce3"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.3. MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3</span></a></h3>
+				<h3 class="link"><a href="#sysscene3"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.3. MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3</span></a></h3>
-                                 <h3 class="link"><a href="#5-sce4"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.4. AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene4</span></a></h3>
+                                 <h3 class="link"><a href="#sysscene4"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.4. AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene4</span></a></h3>
                                  <h3 class="link"><a href="#5-sml"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;5.5. the simulation related to the story</span></a></h3>
 				<h3 class="link"><a href="#5-str"><span style="font-size: 14px;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5.5.1. When the Prince comes?</span></a></h3>
@@ Line 164: / Line 164: @@
 			</div><!-- /_header -->
 			<div id="introduction_contents" class="container_contents">
-				<p class="normal_text">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. </p>
+				<p class="normal_text">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.</p>
-				<p class="normal_text">Most of the stories that have been told for generation have lessons in it. The lessons from Snow White story are mainly the following two points.</p>
 				<p class="normal_text">&#8544;. Do not show off or envy someone.</p>
 				<p class="normal_text">&#8545;. Do not trust unfamiliar people blindly. </p>
-				<p class="normal_text">For a beautiful mind, we need not only to be kind and generous but also to keep a strong heart and control ourselves.</p>
+			<p class="normal_text">	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.</p>
-				<p class="normal_text">In our project, we will recreate Snow White story and evaluate Snow White and the Queen in terms of the beauty inside and out.</p>
@@ Line 177: / Line 174: @@
 			</div><!-- /introduction_contents -->
 		</div><!-- /introduction -->
 		<div id="story" class="container">
 			<div id="story_header" class="container_header">
-				<h2><span>2. Our goal is to evaluate the real beauty of Snow White and the Queen</span></h2>
+				<h2><span>2. Our goal is to evaluate the real beauty of Snow White and the Queen</span></h2><br>
 			</div><!-- /_header -->
 			<div id="story_contents" class="container_contents">
 				<div id="stories">
-				                                <div align="center"><span style="font-style : italic; font-size:18px;">"Magic Mirror on the wall, who is the fairest one of all?"</span></div>
+				                                <div align="center"><span style="font-style : normal_text; font-size:18px;">"Magic Mirror on the wall, who is the fairest one of all?"</span></div><br><br>
-<p class="normal_text">Once upon a time, there lived a Queen.<br>
+       <p class="normal_text">Once upon a time, there lived a Queen.</p><br>
-She was the fairest in the world and she herself also believed so.<br><br>
+ <p class="normal_text">She was the fairest in the world and she herself also believed so.</p><br><br>
-Each time the Queen asked, "Magic Mirror on the wall, who is the fairest one of all?," the mirror would give the same answer.<br>
+ <p class="normal_text">Each time the Queen asked, </p><br>
-"You are the fairest one of all."<br>
+   <p class="normal_text">"Magic Mirror on the wall, who is the fairest one of all?," the mirror would give the same answer.</p><br>
-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.<br><br>
+ <p class="normal_text">"You are the fairest one of all."</p><br><br>
-"Magic Mirror on the wall, who is the fairest one of all?"<br><br>
+ <p class="normal_text">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.</p><br><br>
-One winter night, the Queen asked her mirror as usual.<br>
+ <p class="normal_text">"Magic Mirror on the wall, who is the fairest one of all?"</p><br><br>
-"Magic mirror on the wall, who is the fairest one of all?"<br>
+<p class="normal_text">One winter night, the Queen asked her mirror as usual.</p><br>
-The mirror answered,<br>
+<p class="normal_text">"Magic mirror on the wall, who is the fairest one of all?"</p><br>
-"Snow White is the fairest one."</p><br><br>
+<p class="normal_text">The mirror answered,</p><br>
+<p class="normal_text">"Snow White is the fairest one."</p><br><br>
 				<p class="normal_text">The Snow White was Queen's daughter&#8208;in&#8208;law.</p>
@@ Line 223: / Line 221: @@
-				<p class="normal_text">THE END</p><br><br><br><br>
+				<p class="normal_text">THE END</p><br><br>
 				<div class="gradation_cover"><a href="javascript:void(0);" onclick="show('stories'); return false;"><img src="https://static.igem.org/mediawiki/2016/5/54/T--Tokyo_Tech--readmore.png" /></a></div><!--- /.gradation_cover -->
 				</div><!-- /stories -->
-<p class="normal_text">In our project, we evaluate the real beauty of Snow White and the Queen with the fluorescence intensities of RFP and GFP.
+<p class="normal_text">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.
-  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.
 </p>
-				<p class="normal_text">The REALreal beauty is defined as the followings in this project.<br>
+				<p class="normal_text">The Real beauty is defined as the followings in this project.<br>
 <p class="normal_text">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.  </p>
-                                 <p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Real Beauty =  beauty + inner beauty</span>
-<p class="normal_text">To evaluate the Real beauty, we will recreate Snow White story by using E.coli.</p>
+                                 <p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Real Beauty = outer beauty + inner beauty </span>
+<p class="normal_text">To evaluate the Real beauty, we are going to recreate the Snow White story using <span style="font-style:italic;">E. coli</span>.</p>
 				</p><br>
 				<div align="center"><a href="https://static.igem.org/mediawiki/2016/7/7f/T--Tokyo_Tech--2-1-1.png"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tokyo_Tech--2-1-1.png" height ="500px"></a><br></div>
-				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 2-2-1. </span>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 2-2-1. Time-dependent change of the concentrations of fluorescent protains</span>
 				</p></div><br>
-				<p class="normal_text">  To evaluate the real beauty, we recreated Snow White story by using E.coli.<br>
+				<p class="normal_text">  To evaluate the real beauty, we recreated Snow White story by using <span style="font-style:italic;">E.coli</span>.<br>
 								</p>
@@ Line 253: / Line 251: @@
 			</div><!-- /_header -->
 			<div id="system_contents" class="container_contents">
 				<p class="normal_text">  We will first introduce the  system that forms the basis of our project, the TA system.
                          	</p>
 				<div id="sys1_header">
 					<h3><span>3.1 What is TA system?</span></h3>
 				</div><!-- /_header -->
 				<div id="sys1">
 				<div id="sys1_contents">
-					<p class="normal_text">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.
+					<p class="normal_text">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.
-In general, toxin is more stable than antitoxin, but antitoxin is expressed to a higher level.<br><br>
+<br><br>
                                          <div align="center"><p class="normal_text">First toxin&#8208;antitoxin: MazF / MazE</p></div>
-                                         <p class="normal_text">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. <br>
+                                         <p class="normal_text">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. <br>
-<div align="center"><a href="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--MazMaz.jpeg"><img src="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--MazMaz.jpeg" height ="300"></a><br></div>
+<div align="center"><a href="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--MazMaz.jpeg"><img src="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--MazMaz.jpeg" height ="400"></a><br></div>
-				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 2-2-1. </span>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 2-3-1-1. TA system </span>
 				</p></div><br>
 <br>
@@ Line 272: / Line 270: @@
 					</p>
                                          <div align="center"><p class="normal_text">Second toxin&#8208;antitoxin: YafO / YafN</p></div>
-                                         <p class="normal_text">YafO is a toxin protein, and YafN is its cognate antitoxin protein.  YafO is a ribosome-associated mRNA interferase that cleaves mRNAs’ downstream 11&#8208;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.<br><br>
+                                         <p class="normal_text">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.<br><br>
 					</p>
@@ Line 283: / Line 281: @@
 					<h3><span>3.2 The art of MazF / MazE </span></h3>
 				</div><!-- /_header -->
 				<div id="sys2">
 				<div id="sys2_contents">
-					<p class="normal_text">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. <br>
+					<p class="normal_text">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.<br>
-<p class="normal_text">We will give you an example for a better understanding. Imagine that RFP is expressed little by little in <span style="font-style : italic;">E. coli</span>. <a href="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--Project_RFP.png"><img src="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--Project_RFP.png" width="200px" /></a></p>
+<p class="normal_text">We will give you an example for a better understanding. Imagine that RFP is expressed little by little in <span style="font-style : italic;">E. coli</span>.
-<p class="normal_text">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. <a href="https://static.igem.org/mediawiki/2016/d/d8/T--Tokyo_Tech--Project_MazFE.png"><img src="https://static.igem.org/mediawiki/2016/d/d8/T--Tokyo_Tech--Project_MazFE.png" width="200px" /></a></p>
+	<div align="center"><a href="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--Project_RFP.png"></div>
-<p class="normal_text">In this way, we can control protein translation. That's why we decided to represent Snow White story. <a href="https://static.igem.org/mediawiki/2016/a/aa/T--Tokyo_Tech--Project_MazF_GFP.png"><img src="https://static.igem.org/mediawiki/2016/a/aa/T--Tokyo_Tech--Project_MazF_GFP.png" width="200px" /></a>
+		<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 2-3-2-2 </span>
-					</p>
+		</p></div><br>
+		<br><div align="center"><img src="https://static.igem.org/mediawiki/2016/5/5d/T--Tokyo_Tech--Project_RFP.png" width="200px" /></a></p></div>
+		<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 2-3-2-3</span>
+		</p></div><br>
+		<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 2-3-2-1 </span>
+		</p></div><br>
+<p class="normal_text">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.
+	<br>
+	<div align="center"><a href="https://static.igem.org/mediawiki/2016/d/d8/T--Tokyo_Tech--Project_MazFE.png"><img src="https://static.igem.org/mediawiki/2016/d/d8/T--Tokyo_Tech--Project_MazFE.png" width="200px" /></a></p></div>
+<p class="normal_text">In this way, we can control protein translation. That's why we decided to represent Snow White story. <div align="center"><a href="https://static.igem.org/mediawiki/2016/a/aa/T--Tokyo_Tech--Project_MazF_GFP.png"><img src="https://static.igem.org/mediawiki/2016/a/aa/T--Tokyo_Tech--Project_MazF_GFP.png" width="200px" /></a>
+</p></div>
 				</div>
 				</div>
@@ Line 303: / Line 313: @@
 				<p class="normal_text">Next, we will introduce the genetic circuits that we have designed.
 <p class="normal_text">Our project begins from the scene where the Magic Mirror answers the Queen's question.</p>
 <p class="normal_text">We will split the story into 4 scenes and introduce how our genetic circuits work in each scene. </p><br><br>
@@ Line 310: / Line 321: @@
 					<h3><span>4.1 Scene1 : The magic mirror’s answer</span></h3>
 				</div><!-- /_header -->
 				<div id="sce1">
 				<div id="sce1_contents">
 					<div style="float: left;"><a href="https://static.igem.org/mediawiki/2016/f/f5/T--Tokyo_Tech--koma1.png"><img src="https://static.igem.org/mediawiki/2016/f/f5/T--Tokyo_Tech--koma1.png" height ="200"></a><br></div>
 					<div style="float:left; width: 400px; "><p class="normal_text" style="float: left; margin-left: 15px;">
   <span style="color:#4169e1;">"Magic mirror on the wall, who is the fairest one of all?"<br>
@@ Line 321: / Line 332: @@
 					</p></div>
 					<p class="clear"></p>
-					<div style="text-align: center;  margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/a/a0/T--Tokyo_Tech--Project_Queen_Mirror.png"><img src="https://static.igem.org/mediawiki/2016/a/a0/T--Tokyo_Tech--Project_Queen_Mirror.png" width="400px" /></a>< br />
+					<div style="text-align: center;  margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/a/a0/T--Tokyo_Tech--Project_Queen_Mirror.png"><img src="https://static.igem.org/mediawiki/2016/a/a0/T--Tokyo_Tech--Project_Queen_Mirror.png" width="450px" /></a>
 					<p class="caption"></p></div>
@@ Line 334: / Line 345: @@
 					<h3><span>4.2 Scene2 : The Queen’s trap</span></h3>
 				</div><!-- /_header -->
 				<div id="sce2">
 				<div id="sce2_contents">
 					<div style="float: left;"><a href="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"></a><br></div>
@@ Line 342: / Line 353: @@
 					<p class="clear"></p>
 					<div style="text-align: center;  margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/0/0a/T--Tokyo_Tech--Project_Queen_And_Mirror.png"><img src="https://static.igem.org/mediawiki/2016/0/0a/T--Tokyo_Tech--Project_Queen_And_Mirror.png" width="400px" /></a></div>
 <br>
@@ Line 350: / Line 361: @@
 				<div id="sce3_header">
 					<h3><span>4.3 Scene3 : Snow White's sleep</span></h3>
 				</div><!-- /_header -->
 				<div id="sce3">
 				<div id="sce3_contents">
 					<div style="float: left;"><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Tokyo_Tech--koma3.png" height ="200"><br></div>
@@ Line 364: / Line 375: @@
 					<p class="clear"></p>
-					<div style="text-align:center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png"><img src="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png" width="400px" /></a></div>
+					<div style="text-align:center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png"><img src="https://static.igem.org/mediawiki/2016/a/a5/T--Tokyo_Tech--Project_Queen_And_Snow_White.png" width="600px" /></a></div>
 <p class="normal_text">The basic design of Snow White <span style="font-style: italic;">coli</span>'s genetic circuit is almost the same as the Queen <span style="font-style: italic;">coli</span>'.
-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 <span style="font-style: italic;">coli</span> receives C4HSL and monitor Snow White coli through it. MazF, on the other hand, inhibits the translation in Snow White <span style="font-style: italic;">coli</span>.
+Snow White <span style="font-style:italic;">coli</span> receives 3OC12HSL, the Poisoned Apple, and expresses RhlI and MazF. RhlI synthesizes signaling molecule C4HSL. As described  in the previous section, the Queen <span style="font-style: italic;">coli</span> receives C4HSL and monitor Snow White<span style="font-style:italic;"> coli</span> through it. MazF, on the other hand, inhibits the translation in Snow White <span style="font-style: italic;">coli</span>.
 <br>
@@ Line 377: / Line 388: @@
 					<h3><span>4.4. Scene4 : The Prince’s rescue</span></h3>
 				</div><!-- /_header -->
 				<div id="sce4">
 				<div id="sce4_contents">
-					<div style="float: left;"><a href="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png"><img src="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png" height ="200"></a><br></div>
+					<div style="float: left;"><a href="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png><img src="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png" height ="300"></a><br></div>
 					<div style="float: left; width: 400px;"><p class="normal_text">-----<br>
 <font color="#4169e1">Although he knew her death, he couldn’t help lifting her up because of her beauty.<br>
@@ Line 387: / Line 398: @@
 				<p class="clear"></p>
-				<div style="text-align: center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png"><img src="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png" width="400px" /></a></div>
+				<div style="text-align: center; margin-top: 10px;"><a href="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png"height="100"><img src="https://static.igem.org/mediawiki/2016/7/70/T--Tokyo_Tech--Project_Snow_White_Prince.png" width="400px" /></a></div>
-				<p class="normal_text">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 <span style="font-style: italic;">coli</span>.</p>
+				<p class="normal_text">The Prince <span style="font-style:italic;">coli</span> 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 <span style="font-style: italic;">coli</span>.</p>
 				</div>
 				</div>
@@ Line 405: / Line 416: @@
 					<h3><span>5.1 Cold inducible promoter functions at 18°C in Scene 1</span></h3>
 				</div><!-- /_header -->
-				<div id="sys1">
+				<div id="sysscene1">
-				<div id="sys1_contents">
+				<div id="sysscene1_contents">
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f5/T--Tokyo_Tech--koma1.png" height ="200"></div><br>
+                                <p class="normal_text">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.</p>
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/0d/Tokyo_Tech_cold.png" height ="300"></div>
-                                 <p class="normal_text">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 18°C had higher fluorescence intensity of GFP than those cultured at 37°C.</p>
+                                 <p class="normal_text">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.</p>
-                                 <p class="normal_text">Thus, we found that the story begins with lowering the culture temperature .</p>
+                                 <p class="normal_text"> Thus, it has been confirmed that the story could be begun with lowering the culture temperatures </p>
+<a href="https://2016.igem.org/Team:Tokyo_Tech/Promoter_Assay/Pcold ">Read Pcold Assay page </a>.
 <br><br></p>
-  				<p class="normal_text">5.2 The screening of Prhl with optimal strength for Scene 2<br>
+  				        <h3><span>5.2 The screening of Prhl with optimal strength for Scene 2</span><h3>
-  				<p class="normal_text">Eating the Poisoned Apple given by the Queen <span style="font-style : italic">coli</span>, Snow White <span style="font-style : italic">coli</span> 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.</p>
+                                </div><!-- /_header -->
-<p class="normal_text">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. </p>
+				<div id="sysscene2">
+				<div id="sysscene2_contents">
+					<div align="center"><a href="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"></a><br></div>
+  				<p class="normal_text">Eating the Poisoned Apple given by the Queen <span style="font-style : italic">coli</span>, the Snow White <span style="font-style : italic">coli</span> 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.</p>
+<p class="normal_text">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. </p>
 <p class="normal_text">  5.2.1　Reporter assay<br>
-<p class="normal_text">First, we evaluated the activities the existing promoters: Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) by adding 3 types of AHLs.
+                                </div><!-- /_header -->
+                                <div id="ass">
+				<div id="ass_contents">
+<p class="normal_text">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).
 					</p>
 					<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/00/T--Tokyo_Tech--assay_ahl_reporter.png" height ="200"></div>
-					<p class="normal_text">Even adding 3OC12HSL to Plux promoter, the promoter activity was still observed. The graph also shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when C4HSL is added.</p>
+					<p class="normal_text"> 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.</p>
+<a href="https://2016.igem.org/Team:Tokyo_Tech/AHL_Assay/AHL_Reporter_Assay
+ ">Read AHL Reporter Assay</a>.
 				<br>
@@ Line 427: / Line 456: @@
 				<p class="normal_text">  5.2.2 Simulation regarding Prhl strength　<br>
-<p class="normal_text">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.<br>
+                                </div><!-- /_header -->
+                                <div id="sml">
+				<div id="sml_contents">
+<p class="normal_text"> 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.<br>
+	<div align="center"><img src="https://static.igem.org/mediawiki/2016/6/68/T--Tokyo_Tech--projct_model1.png" height ="300"><br></div>
 <p class="normal_text">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.</p>
-<p class="normal_text">The graph below shows the result of recreating “Snow White” when using existing rhl promoter. Only the fluorescence intensity of the Queen coli got lowered. It means that the Queen coli failed to poison Snow White coli and she killed herself.</p>
+<a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prhl ">Read Model page</a>.
 			</p><br>
 					</p><br>
 				<p class="normal_text">  5.2.3　Improvement of Prhl<br>
-We made mutants of Prhl by inserting a point mutation into the wild type (WT) Prhl.
+                                </div><!-- /_header -->
-In the experiment, we added reagent AHLs into the reporters and examined their fluorescence intensities.
+                                <div id="imp">
-As a result, we succeeded in newly obtaining stronger mutants than the WT Prhl (NM).<br>
+				<div id="imp_contents">
-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 <span style="font-style : italic">coli</span> will suicide by eating the Poisoned Apple made by herself. Therefore, we made the mutants using WT as a template.<br>
+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).<br>
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/e/ef/Tokyo_Tech_Prhlimp.png" height ="300"><br></div>
+<p class="normal_text">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 <span style="font-style : italic">coli</span> 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.</p><a href="https://2016.igem.org/Team:Tokyo_Tech/AHL_Assay/Rhl_System_Assay ">Read Rhl System Assay </a>.<br>
 								</p><br>
-			<div id="sce2_header">
 					<h3><span>5.3 MazF-MazE system as Toxin-Antitoxin system can be controlled in Scene 3</span></h3>
 				</div><!-- /_header -->
+                                <div id="sysscene3">
+				<div id="sysscene3_contents">
+					<div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Tokyo_Tech--koma3.png" height ="200"></div>
 <p class="normal_text">In this Scene, we introduce TA system as a concept.</p>
 <p class="normal_text">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.”</p>
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/42/T--Tokyo_Tech--maz1.png" height ="300"></div>
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tokyo_Tech--maz2.png" height ="300"></div>
 <p class="normal_text">In this experiment, we used the BBa_K1949100 and Bba_1949102.</p>
 <p class="normal_text">First, MazF was expressed by arabinose, and 2 h later, the MazE was expressed by IPTG/p.
@@ Line 451: / Line 494: @@
 Moreover, when only MazF worksed, the RFU of GFP hardly rose, but when MazE was induced, the RFU of GFP rose. (link: toxin assay)<br>
 </p><p class="normal_text">From the above, we found that MazF stops cell growth and translation of <span style="font-style : italic">E. coli</span>, but MazE restarts cell growth that have stopped and furthermore restarts translation.</p>
-According to the experiments, we showed that TA system works properly.</p>                                                                     </p><br>
+<p class="normal_text">According to the experiments, we showed that TA system works properly.</p> <a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay/mazEF_System_Assay
+ ">Read mazEF System Assay</a>.
+                                                                     </p><br>
+  				        <h3><span>5.4 AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene 4</span></h3></span>
+                                </div><!-- /_header -->
+                                <div id="sysscene4">
+				<div id="sysscene4_contents">
-                                                              <p class="normal_text">5.4 AmiE degrades 3OC12HSl selectively and does not degrade C4HSL in Scene 4<br>
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png" height ="200"></div>
-In the final Scene, we introduce the selective degradation of AHLs by AmiE as a concept.
+<p class="normal_text">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 <span style="font-style : italic">coli</span>. Therefore, we examined whether AmiE selectively degrades AHLs.<br>
 From the experimental results, when C12 was added to the culture skution of <span style="font-style : italic">E. coli</span> 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.<br>
+For these reasons, we showed that AmiE selectively degrades AHLs, only C12 in this project.<br></p>
+<a href="https://2016.igem.org/Team:Tokyo_Tech/AmiE_Assay
+ ">Read AmiE Assay</a>.
                                                                      </p><br>
-                                                              <p class="normal_text">5.5　the simulation related to the story<br>
+                                         <h3><span>5.5　the simulation related to the story</span></h3>
+                                </div><!-- /_header -->
+                                <div id="5-sml">
+				<div id="5-sml_contents">
+ <p class="normal_text">
 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.<br>
                                                                      </p><br>
                                                                <p class="normal_text">5.5.1. When the Prince comes?<br>
 We simulated to confirm which is better, the Prince <span style="font-style : italic">coli</span> 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 <span style="font-style : italic">coli</span> is added from the beginning, AmiE produced by the Prince <span style="font-style : italic">coli</span> increases and C12 is overdegraded. Then, C12 cannot exist in the medium (Fig. 2), and the circuit does not work correctly.<br>
+				    <div align="center"><img src="https://static.igem.org/mediawiki/2016/1/1f/T--Tokyo_Tech--Population-miss.png" height ="300" ></div>
+	<div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f1/T--Tokyo_Tech--AHL-miss.png" height ="300" class="align_right"></div>
+                   <p class="normal_text">As a result, if we introduce the Prince <span style ="font-style : italic">coli</span> from the beginning, the number of Prince <span style ="font-style : italic">coli</span> increases too much (Fig.5-4-1-1) so the AmiE the Prince <span style ="font-style : italic">coli</span> produces augments and the decomposition of C12 also occurs overly. So C12 is almost inexistent in the medium (Fig.5-4-1-2).</p>
+<p class="normal_text">On the other hand, when the Prince <span style="font-style : italic">coli</span> is added at t = 700, the number of the Prince <span style="font-style : italic">coli</span> does not increase greatly (Fig3). Therefore, C12 can exist until t = 70, and after that C12 decreases with an increase in AmiE (Fig4).<br></p>
+                 <div align="center"><img src="https://static.igem.org/mediawiki/2016/e/e7/T--Tokyo_Tech--Population.png" height ="300" class="align_left"></div>
+				    <div align="center"><img src="https://static.igem.org/mediawiki/2016/4/4f/T--Tokyo_Tech--AHL.png" height ="300" class="align_right"></div>
+<p class="normal_text">From this result, it was found that the genetic circuit works well by adding the Prince <span style="font-style : italic">coli</span> at t=700. In other words, as with the original story, the Prince <span style="font-style : italic">coli</span> comes across Snow White <span style="font-style : italic">coli</span> at t=700 and rescues her.<br></p><a href="https://2016.igem.org/Team:Tokyo_Tech/Model#prince_coli ">Read Model page</a>.
+				</div><!-- /_header -->
+                                <div id="5-str">
+				<div id="5-str_contents">
-On the other hand, when the Prince <span style="font-style : italic">coli</span> is added at t = 700, the number of the Prince <span style="font-style : italic">coli</span> does not increase greatly (Fig3). Therefore, C12 can exist until t = 70, and after that C12 decreases with an increase in AmiE (Fig4).<br>
-From this result, it was found that the genetic circuit works well by adding the Prince <span style="font-style : italic">coli</span> at t=700. In other words, as with the original story, the Prince <span style="font-style : italic">coli</span> comes across Snow White <span style="font-style : italic">coli</span> at t=700 and rescues her.<br>
                                                                      </p><br>
                                                                <p class="normal_text">5.5.2　representation of the story<br>
+                                </div><!-- /_header -->
+                                <div id="5-str2">
+				<div id="5-str2_contents">
 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.<br>
 Based on the simulation, we will show you which one is a real beauty, Snow White <span style="font-style : italic">coli</span> or the Queen <span style="font-style : italic">coli</span>.<br>
+<div class="floating"><img src="https://static.igem.org/mediawiki/2016/7/79/T--Tokyo_Tech--4koma.png" width ="320"class="align_right"> </div>
+     <p class="normal_text"> In the blue area of Fig.5-2-2, the concentration of fluorescent proteins start to increase. The concentration of RFP of Snow White <span style ="font-style : italic">coli</span> exceeds  that of GFP of the Queen <span style ="font-style : italic">coli</span>. <br>
+     It is as if Snow White got fairer more and more.<br><br>
+     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 <span style ="font-style : italic">coli</span> and the Queen <span style ="font-style : italic">coli</span> start to suppress the increment of fluorescet proteins. <br>
+     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.<br><br>
+     In the green area of Fig.5-2-2, the concentration of C12 more increases and the MazF inside Snow White <span style ="font-style : italic">coli</span> more suppress the increment of GFP. So the concentration GFP exceeds that of RFP. <br>
+     It looks as if Snow White bit the apple, sinking into unconsciousness soon.<br><br>
+     In the yellow area of Fig.5-2-2, the AmiE synthesized by the introduced Prince <span style ="font-style : italic">coli</span> decomposes C12 so the MazF inside Snow White <span style ="font-style : italic">coli</span> diminishes and the concentration of GFP resumes. <br>
+     It looks as if the Prince lifted Snow White and she opened her eyes.
-In the blue area of Fig, the fluorescence intensity of Snow White <span style="font-style : italic">coli</span> exceeds the Queen <span style="font-style : italic">coli</span>’s.
-That is because one day, Snow White <span style="font-style : italic">coli</span>’s beauty exceeded the Queen <span style="font-style : italic">coli</span>’s beauty. Snow White got fairer and fairer.<br>
-In the pink area of Fig, added C4 leads to production of C12. Because of this, MazF, a toxin, increases in the Queen <span style="font-style : italic">coli</span>, 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<br>
-In the green area of Fig, incereased C12 induces MazF in Snow White <span style="font-style : italic">coli</span>, which causes GFP excess over RFP. It looks as if Snow White bit the apple, then sank into unconsciousness soon<br>
-In the yellow area of Fig, since AmiE produced by the added Prince <span style="font-style : italic">coli</span> degrades C12, MazF in Snow White <span style="font-style : italic">coli</span> decreases and increases C4. It looks as if the Prince lifted Snow White and she opened her eyes.<br>
-From the above, we were able to represent the story by the genetic circuit we designed, and furthermore, it was found that Snow White <span style="font-style : italic">coli</span> is a real beauty. <br>
                                                                      </p>
@@ Line 497: / Line 576: @@
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 </div></div>
+<!-- Additional --></div>
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@@ Line 503: / Line 583: @@
 			</div><!-- /_header -->
 			<div id="hp_contents" class="container_contents">
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/2/2a/Tokyo_Tech_HP.png" height ="300"><br></div>
 				<p class="normal_text"> 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.
 <br>
                          	</p><br><br>
 				<div id="cha1_header">
 					<h3><span>6.1 “Snow White”</span></h3>
 				</div><!-- /_header -->
 				<div id="cha1">
 				<div id="cha1_contents">
 					<p class="normal_text">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.<br>
 					<h3><span>6.2 Addition of other characters</span></h3>
 				</div><!-- /_header -->
 				<div id="cha21">
 				<div id="cha2_contents">
 					<p class="normal_text">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 <span style="font-style : italic">E. coli</span> (strain name). As you can see in the photograph, they have round shapes and are very charming compared to the general <span style="font-style : italic">E. coli</span>. <br><br></div></div><!-- /cha21 -->
@@ Line 523: / Line 604: @@
 					<h3><span>6.3. The software development for future work</span></h3>
 				</div><!-- /_header -->
 				<div id="sof">
 				<div id="sof_contents">
 					<p class="normal_text">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.<br><br>
@@ Line 530: / Line 611: @@
 					<h4><span>6.3.1 The dialogue with an expert</span></h4>
 				</div><!-- /_header -->
 				<div id="dia">
 				<div id="dia_contents">
 					<p class="normal_text">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.</p>
 <br><br>
 					</p></div>
                                  <div id="dwa_header">
 					<h4><span>6.3.2 ACA Dwarfs</span></h4>
 				</div><!-- /_header -->
 				<div id="dwa">
 				<div id="dwa_contents">
+<div align="center"><img src="https://static.igem.org/mediawiki/2016/8/8b/T--Tokyo_Tech--ACA_Dwarfs.jpg" height ="300"><br></div>
 					<p class="normal_text">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.<br>
@@ Line 546: / Line 628: @@
 Then, our dry lab used Java to develop software named “ACA Dwarfs” adjusting number of ACA base sequence as a solution to the problem.</p>
 <br>
-  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.<br>
-Using ACADwarfs enabled us to make GFP and RFP more reactive to MazF.<br>
@@ Line 566: / Line 646: @@
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 				<p class="normal_text">[1] Unterholzner SJ, Poppenberger B, Rozhon W. Toxin-antitoxin systems: Biology, identification, and ampicillin. Mob Genet Elements. 2013 Sep; 3(5): e26219.</p>
 <p class="normal_text">[2]Zielenkiewicz U., Ceglowski P. The toxin-antitoxin system of the streptococcal plasmid pSM19035.J. Bacteriol. 2005;187:6094–6105.</p>
 <p class="normal_text">[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.</p>
 <p class="normal_text">[4] Gerdes K, Wagner EG. RNA antitoxins. Curr. Opin. Microbiol. 2007 Apr; 10 (2): 117–24. </p>
 <p class="normal_text">[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.</p>
 <p class="normal_text">[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.</p>
 <p class="normal_text">[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. </p>
 <p class="normal_text">[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.</p>
 <p class="normal_text">[9] Zhang Y, Yamaguchi Y, Inouye M. Characterization of YafO, an Escherichia coli toxin. J Biol Chem. 2009 Sep; 284(38): 25522–25531.</p>
 <p class="normal_text">[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. </p>
 <p class="normal_text">[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.</p>
 <p class="normal_text">
 [12] Gerardo Medina et al. Mechanism of Pseudomonas aeruginosa RhlR Transcriptional Regulation of the rhlAB Promoter. J Bacteriol. 2003 Oct; 185(20): 5976–5983.</p>
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