Revision as of 06:15, 18 October 2016

Model

1. Overview

To reproduce the story of ”Snow White”, we have designed the cell-cell communication system with improved or characterized parts and collected data from comprehensive experiments. Furthermore, we constructed the mathematical model in order to simulate the behavior of the whole system and to confirm the feasibility of our story. This simulation successfully contributed to give the suggestions to wet lab experiments and confirm the feasibility of our system. In addition, in order to utilize TA (Toxin-Antitoxin) system, we developed a new software in Java for adjusting the number of ACA sequences, which MazF dimer recognizes and cleaves in mRNAs.

2. Story Simulation

2-1. Mathematical Model

To simulate our gene circuits, we developed an ordinary differential equation model.

[Detailed Description for Modeling]

Differencial Equations

Snow White

\begin{equation} \frac{d[mRNA_{RFP}]}{dt} = k - d[mRNA_{RFP}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{RFP}}})[mRNA_{RFP}][DiMazF] \end{equation} \begin{equation} \frac{d[mRNA_{RhlI}]}{dt} = leak_{P_{lux}} + \frac{\kappa_{Lux}[C12]^{n_{Lux}}}{K_{mLux}^{n_{Lux}} + [C12]^{n_{Lux}}} - d[mRNA_{RhlI}] - F_{DiMazF}f[mRNA_{RhlI}][DiMazF] \end{equation} \begin{equation} \frac{d[RFP]}{dt} = \alpha [mRNA_{RFP}] - d_{RFP}[RFP] \end{equation} \begin{equation} \frac{d[RhlI]}{dt} = \alpha [mRNA_{RhlI}] - d_{RhlI}[RhlI] \end{equation} \begin{equation} \frac{d[C4]}{dt} = p_{C4}[RhlI]P_{Snow White} - d_{C4}[C4] \end{equation} \begin{equation} \frac{d[mRNA_{MazF}]}{dt} = leak_{P_{lux}} + \frac{\kappa_{Lux}[C12]^{n_{Lux}}}{K_{mLux}^{n_{Lux}}+ [C12]^{n_{Lux}}} \\ 　　　　　　　- d[mRNA_{MazF}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazF}}})[mRNA_{MazF}][DiMazF] \end{equation} \begin{equation} \frac{d[mRNA_{MazE}]}{dt} = k - d[mRNA_{MazE}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazE}}})[mRNA_{MazE}][DiMazF] \end{equation} \begin{equation} \frac{d[MazF]}{dt} = \alpha [mRNA_{MazF}] - 2k_{Di_{MazF}}[MazF] + 2k_{-Di_{MazF}}[DiMazF] - d_{MazF}[MazF] \end{equation} \begin{equation} \frac{d[DiMazF]}{dt} = k_{Di_{MazF}}[MazF] - k_{-Di_{MazF}}[DiMazF] - 2k_{Hexa}[DiMazE][DiMazF]^2 \\ 　　　　　　　+ 2k_{-Hexa}[MazHexamer] - d_{DiMazF}[DiMazF] \end{equation} \begin{equation} \frac{d[MazE]}{dt} = \alpha [mRNA_{MazE}] - 2k_{Di_{MazE}}[MazE] + 2k_{-Di_{MazE}}[DiMazE] - d_{MazE}[MazE] \end{equation} \begin{equation} \frac{d[DiMazE]}{dt} = k_{Di_{MazE}}[MazE] - k_{-Di_{MazE}}[DiMazE] - k_{Hexa}[DiMazE][DiMazF]^2 \\ 　　　　　　　+ k_{-Hexa}[MazHexamer] - d_{DiMazE}[DiMazE] \end{equation} \begin{equation} \frac{d[MazHexa]}{dt} = k_{Hexa}[DiMazE][DiMazF]^2 - k_{-Hexa}[MazHexa] - d_{Hexa}[MazHexa] \end{equation} \begin{equation} \frac{dP_{Snow White}}{dt} = g \frac{E_{DiMazF}}{E_{DiMazF}+[DiMazF]}\left(1- \frac{P_{Snow White}+P_{Queen}+P_{Prince}}{P_{max}} \right) P_{Snow White} \end{equation}

Queen

\begin{equation} \frac{d[mRNA_{GFP}]}{dt} = k - d[mRNA_{GFP}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{GFP}}})[mRNA_{GFP}][DiMazF] \end{equation} \begin{equation} \frac{d[mRNA_{LasI}]}{dt} = leak_{P_{rhl}} + \frac{\kappa_{Rhl}[C4]^{n_{Rhl}}}{K_{mRhl}^{n_{Rhl}} + [C4]^{n_{Rhl}}} \\ 　　　　　　　- d[mRNA_{LasI}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{LasI}}})[mRNA_{LasI}][DiMazF] \end{equation} \begin{equation} \frac{d[GFP]}{dt} = \alpha [mRNA_{GFP}] - d_{GFP}[GFP] \end{equation} \begin{equation} \frac{d[LasI]}{dt} = \alpha [mRNA_{LasI}] - d_{LasI}[LasI] \end{equation} \begin{equation} \frac{d[C12]}{dt} = p_{C12}[LasI]P_{Queen} - d_{C12}[C12] - D[C12][AmiE] \end{equation} \begin{equation} \frac{d[mRNA_{MazF}]}{dt} = leak_{P_{lux}} + \frac{\kappa_{Rhl}[C4]^{n_{Rhl}}}{K_{mRhl}^{n_{Rhl}} + [C4]^{n_{Rhl}}} \\ 　　　　　　　- d[mRNA_{MazF}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazF}}})[mRNA_{MazF}][DiMazF] \end{equation} \begin{equation} \frac{d[mRNA_{MazE}]}{dt} = k - d[mRNA_{MazE}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazE}}})[mRNA_{MazE}][DiMazF] \end{equation} \begin{equation} \frac{d[MazF]}{dt} = \alpha [mRNA_{MazF}] - 2k_{Di_{MazF}}[MazF] + 2k_{-Di_{MazF}}[DiMazF] - d_{MazF}[MazF] \end{equation} \begin{equation} \frac{d[DiMazF]}{dt} = k_{Di_{MazF}}[MazF] - k_{-Di_{MazF}}[DiMazF] - 2k_{Hexa}[DiMazE][DiMazF]^2 \\ 　　　　　　　+ 2k_{-Hexa}[MazHexamer] - d_{DiMazF}[DiMazF] \end{equation} \begin{equation} \frac{d[MazE]}{dt} = \alpha [mRNA_{MazE}] - 2k_{Di_{MazE}}[MazE] + 2k_{-Di_{MazE}}[DiMazE] - d_{MazE}[MazE] \end{equation} \begin{equation} \frac{d[DiMazE]}{dt} = k_{Di_{MazE}}[MazE] - k_{-Di_{MazE}}[DiMazE] - k_{Hexa}[DiMazE][DiMazF]^2 \\ 　　　　　　　+ k_{-Hexa}[MazHexamer] - d_{DiMazE}[DiMazE] \end{equation} \begin{equation} \frac{d[MazHexa]}{dt} = k_{Hexa}[DiMazE][DiMazF]^2 - k_{-Hexa}[MazHexa] - d_{Hexa}[MazHexa] \end{equation} \begin{equation} \frac{dP_{Queen}}{dt} = g \frac{E_{DiMazF}}{E_{DiMazF}+[DiMazF]}\left(1- \frac{P_{Snow White}+P_{Queen}+P_{Prince}}{P_{max}}\right) P_{Queen}\\ \end{equation}

Prince

\begin{equation} \frac{d[mRNA_{AmiE}]}{dt} = leak_{P_{lux}} + \frac{\kappa_{Lux}[C12]^n}{K_{mLux}^n + [C12]^n} - d[mRNA_{AmiE}] \end{equation} \begin{equation} \frac{d[AmiE]}{dt} = \alpha [mRNA_{AmiE}]P_{Prince} - d_{AmiE}[AmiE] \end{equation} \begin{equation} \frac{dP_{Prince}}{dt} = g\left(1- \frac{P_{Snow White}+P_{Queen}+P_{Prince}}{P_{max}}\right) P_{Prince} \end{equation}

Explanation about Parameters

Parameter	Description
$$g$$	Growth rate of each cells
$$P_{max}$$	Carrying capacity
$$E_{DiMazF}$$	Effect of MazF dimer on growth rate
$$k$$	Transcription rate of mRNA under $P_{tet}$
$$leak_{P_{lux}}$$	Leakage of $P_{lux}$
$$leak_{P_{rhl}}$$	Leakage of $P_{rhl}$
$$\kappa_{Lux}$$	Maximum transcription rate of mRNA under $P_{lux}$
$$\kappa_{Rhl}$$	Maximum transcription rate of mRNA under $P_{rhl}$
$$n_{Lux}$$	Hill coefficient for $P_{lux}$
$$n_{Rhl}$$	Hill coefficient for $P_{rhl}$
$$K_{mLux}$$	Lumped paremeter for the Lux System
$$K_{mRhl}$$	Lumped paremeter for the Rhl System
$$F_{DiMazF}$$	Cutting rate at ACA sequences on mRNA by MazF dimer
$$f$$	The probability of distinction of ACA sequencess in each mRNA
$$f_{mRNA_{RFP}}$$	The number of ACA sequences in $mRNA_{RFP}$
$$f_{mRNA_{GFP}}$$	The number of ACA sequences in $mRNA_{GFP}$
$$f_{mRNA_{RhlI}}$$	The number of ACA sequences in $mRNA_{RhlI}$
$$f_{mRNA_{LasI}}$$	The number of ACA sequences in $mRNA_{LasI}$
$$f_{mRNA_{MazF}}$$	The number of ACA sequences in $mRNA_{MazF}$
$$f_{mRNA_{MazE}}$$	The number of ACA sequences in $mRNA_{MazE}$
$$\alpha$$	Translation rate of Protein
$$k_{Di_{MazF}}$$	Formation rate of MazF dimer
$$k_{-Di_{MazF}}$$	Dissociation rate of MazF dimer
$$k_{Di_{MazE}}$$	Formation rate of MazE dimer
$$k_{-Di_{MazE}}$$	Dissociation rate of MazE dimer
$$k_{Hexa}$$	Formation rate of Maz hexamer
$$k_{-Hexa}$$	Dissociation rate of Maz hexamer
$$p_{C4}$$	Production rate of C4HSL by RhlI
$$p_{C12}$$	Production rate of 3OC12HSL by LuxI
$$D$$	Decomposition rate of 3OC12HSL by AmiE
$$d$$	Degradation rate of mRNA
$$d_{RFP}$$	Degradation rate of RFP
$$d_{GFP}$$	Degradation rate of GFP
$$d_{RhlI}$$	Degradation rate of RhlI
$$d_{LasI}$$	Degradation rate of LasI
$$d_{MazF}$$	Degradation rate of MazF
$$d_{DiMazF}$$	Degradation rate of MazF dimer
$$d_{MazE}$$	Degradation rate of MazE
$$d_{DiMazE}$$	Degradation rate of MazE dimer
$$d_{Hexa}$$	Degradation rate of Maz Hexamer
$$d_{C4}$$	Degradation rate of C4HSL
$$d_{C12}$$	Degradation rate of 3OC12HSL
$$d_{AmiE}$$	Degradation rate of AmiE

2-2. Results

As a result, we obtained and confirmed the desirable behavior of the whole system by modifying and improving parts. As described below, our simulation showed appropriate transition of fluorescence for the story.

Fig. 5-1.Time-dependent change of the concentrations of fluorescence proteins

Fig. 5-2.The story of "Snow White"

In the blue area of Fig, the fluorescence intensity starts to increase. Snow White coli’s fluorescence intensity exceeds that of the Queen coli’s. It represents Snow White got fairer more and more.
In the pink area of Fig, C12 is being synthesized thanks to the C4’s appearance. And the concentration of C12 increases. As a result, the toxin MazF augments inside of Snow White coli and the Queen coli, suppressing the increment of fluorescent proteins. It is as if the Mirror’s answer transformed the Queen into a Witch, so she can give Snow White a poisoned apple.
In the green area of Fig, C12 increases and the MazF inside Snow White coli induced by it increases even more. So the GFP exceeds the RFP. It looks as if Snow White bit the apple, sinking into unconsciousness promptly.
In the yellow area of Fig, the AmiE synthesized by the introduced Prince coli decomposes the C12 molecules so the amount of MazF inside Snow White diminishes and the amount of C4 increases. It looks as if Prince lifted Snow White and she opened her eyes.

3. Fitting

TEST

4. Sensitivity Analysis

We performed the sensitivity analysis descried in this section in order to examine which parameter dominates the story.

4-1. Requirements

We defined these requirements as the “successful Snow White story.”
1) At t = 150
concentration of RFP > concentration of GFP
2) At t = 700
concentration of RFP< concentration of GFP
3) At t = 1500
concentration of RFP > concentration of GFP
We the began to analyze the graph that satisfies these requirements that we could say that reproduces the story correctly. In the table bellow we show the range in which we modified each parameter. They were modified one step size at a time.

Parameter	Range	Step size
$$D$$	$$0.0001	$$0.0001$$
$$p_{C4}$$	$$0.0001	$$0.001$$
$$p_{C12}$$	$$0.0001	$$0.001$$
$$α$$	$$0.01<α<0.2$$	$$0.01$$
$$d_{AmiE}$$	$$0.001	$$0.001$$

As a result, we obtained the following parameter ranges.

Parameter	Value
$$D$$	$$0.0056
$$p_{C4}$$	$$0.0029
$$p_{C12}$$	$$0.001
$$α$$	$$0.01<α<0.16$$
$$d_{AmiE}$$	$$0.001

4-2. the Prince coli should be put in in the middle

We run simulations in order to determine whether we would get a better behavior let we introduce the Prince coli at the beginning or halfway of the story.

Fig. ??.Number of individuals when the Prince coli is introduced from the beginning

Fig. ??.AHL concentrations when the Prince coli is introduced from the beginning

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

Fig. ??.Number of individuals when the Prince coli is introduced at t = 700

Fig. ??.AHL concentrations when the Prince coli is introduced at t = 700

On the other hand, if we introduce the Prince coli at t=700, the number of Prince coli does not increment much (Fig ), so C12 can exist until t=700 and then decreases thanks to the augment of AmiE (Fig ).
In conclusion, if we introduce the Prince coli at t=700, the circuit will behave accordingly.

4-3. Prhl should be changed

In order to confirm whether by using a combination of the current promoters we would be able to reproduce the story with our gene circuit, we performed some simulations based on the results of our assays.

Fig. The intensity of Plux and Prhl promoters

The diagram above shows that the intensity of the two promoters should be in the red region of the figure.The combination of promoters we were originally going to use is shown in the graph by the green point. To move this point into the red region, we had to improve on Prhl so its expression level increases.

4-4. Production rate of C4HSL and 3OC12HSL by RhlI and LasI

The signaling molecule production rates by RhlI and LasI can be changed by modifying RhlI and LasI to make more or less signaling molecule in silico.

Fig. Concentrations of GFP and RFP dependencies of production rate of C4 by RhlI

Each line corresponds to the transition of the concentration of RFP/GFP with a certain production rate of C12. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher production rate of C12.
if the production rate of C4 is between 0.029 and 0.778, our system can reproduce the story.
If this parameter is too small, The production of LasI by the Queen coli is insufficiently inhibited by MazF so C12 increases greatly. As a result, The concentration of GFP overcomes the concentration of RFP and the story does not develop correctly. And if this parameter is too big, The production of LasI by the Queen coli is overly inhibited by MazF so C12 does not increase. As a result, the concentration of RFP is always greater than the GFP concentration and the story does not develop either.

Fig. Concentrations of GFP and RFP dependencies of production rate of C12 by LasI

Each line corresponds to the transition of the concentration of RFP/GFP with a certain production rate of C4. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher production rate of C4.
If production rate of C12 is between 0.001 and 0.217, our system can reproduce the story.

4-5. Translation rate of protein

Translation rate affects the production of protein.

Fig. Concentrations of GFP and RFP dependencies of translation rate of proteins

Each line corresponds to the transition of the concentration of RFP/GFP with a certain translation rate. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher translation rate.
If translation rate of protein is between 0.01 and 0.16, our system can reproduce the story.

4-6. Decomposition rate of C12 by AmiE

AmiE decomposes C12. The concentration of C12 after input of the Prince coli is changed by AmiE. If the decomposition rate of C12 is too small, C12 does not decrease enough so the MazF inside Snow White coli continues being expressed and the concentration of RFP decreases.

Fig. Concentrations of GFP and RFP dependencies of decomposition rate of C12 by AmiE

Each line corresponds to the transition of the concentration of RFP/GFP with a certain value of D. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher value of decomposition rate of C12.
If degradation rate of C12 is higher than 0.00056, our system can reproduce the story.

4-7. Degradation rate of AmiE

Fig. Concentration of GFP and RFP dependencies of degradation rate of AmiE

Each line corresponds to the transition of the concentration of RFP/GFP with a certain degradation rate of AmiE. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher degradation rate of AmiE.
Even if we modify the values of the parameters inside the defined range, the concentration of RFP overcomes the concentration of GFP. And even if the degradation rate of AmiE is small, the decomposition rate of C12 by AmiE is high enough so C12 decreases sufficiently.

4-8. Degradation rate of RFP and GFP

The degradation rate of RFP and GFP is key to the success the story of ‘Snow White’.
These parameters are closely related to the concentrations of GFP and RFP, so we conjectured that if they do not take appropriate values the story can not be correctly reproduced.

Fig. Relation of degradation rate of GFP and RFP

The story is reproduced only if the degradation rate of RFP and GFP are the same.

5. Software

5-1. Abstract

We developed a new software named ACADwarfs. This software helps to control the sensitivity of the protein to MazF by regulating the number of ACA sequences in the mRNA sequence. ACADwarfs can increase or decrease the number of ACA sequences on mRNA without changing the amino acid sequences that the mRNA specifies or frameshifts resulted from insertion of bases without considering.
Therewith we improve the practicality of the characteristic of the mazEF system. For example you can let protein A express constantly by eliminating ACA sequences of the sequence, while letting protein B suspend to express, at the desirable timing, by expression of MazF.
This software also evades using rare codons, so you don’t have to worry about them.

5-2. Key achievement

・Provided the tool regulating the number of ACA sequences
・Released under open-source license so everyone can use it
・Able to correspond to any base arrangements
・Rare codons are evaded
・Extend the application field of mazE system

5-3. Work flow

TEST

5-4. Demonstration

TEST

5-5. Download

To download click here.

The code is available on github.

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-			<h1 align="center">Project</h1>
+			<h1 align="center">Model</h1>
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-				<h3 class="link"><a href="#introduction">1. Introduction</a></h3>
+				<h3 class="link"><a href="#overview">1. Overview</a></h3>
-				<h3 class="link"><a href="#story">2．Story</a></h3>
+				<h3 class="link"><a href="#story_simulation">2. Story Simulation</a></h3>
-				<h3 class="link"><a href="#design">3．Scene ＆ Design</a></h3>
+				<h3 class="link"><a href="#mathematical_model"><font size="2.7">&nbsp;&nbsp;&nbsp;2-1. Mathematical Model</font></a></h3>
-				<h3 class="link"><a href="#sce1"><font size="2.7">&nbsp;&nbsp;&nbsp;3.1 Scene1</font></a></h3>
+				<h3 class="link"><a href="#results"><font size="2.7">&nbsp;&nbsp;&nbsp;2-2. Results</font></a></h3>
-				<h3 class="link"><a href="#sce2"><font size="2.7">&nbsp;&nbsp;&nbsp;3.2 Scene2</font></a></h3>
+				<h3 class="link"><a href="#fitting">3. Fitting</a></h3>
-				<h3 class="link"><a href="#sce3"><font size="2.7">&nbsp;&nbsp;&nbsp;3.3 Scene3</font></a></h3>
+				<h3 class="link"><a href="#analysis">4. Sensitivity Analysis</a></h3>
-				<h3 class="link"><a href="#sce4"><font size="2.7">&nbsp;&nbsp;&nbsp;3.4 Scene4</font></a></h3>
+				<h3 class="link"><a href="#requirements"><font size="2.7">&nbsp;&nbsp;&nbsp;4-1. Requirements</font></a></h3>
-				<h3 class="link"><a href="#function">4．Function</a></h3>
+				<h3 class="link"><a href="#prince_coli"><font size="2.7">&nbsp;&nbsp;&nbsp;4-2. the Prince <span style ="font-style : italic">coli</span> should be put in in the middle</font></a></h3>
-				<h3 class="link"><a href="#fun1"><font size="2.7">&nbsp;&nbsp;&nbsp;4.1 Scene1</font></a></h3>
+				<h3 class="link"><a href="prhl"><font size="2.7">&nbsp;&nbsp;&nbsp;4-3. Prhl should be changed</font></a></h3>
-				<h3 class="link"><a href="#fun2"><font size="2.7">&nbsp;&nbsp;&nbsp;4.2 Scene2</font></a></h3>
+				<h3 class="link"><a href="production_ahl"><font size="2.7">&nbsp;&nbsp;&nbsp;4-4. Production rate of C4HSL and 3OC12HSL by RhlI and LasI</font></a></h3>
-				<h3 class="link"><a href="#fun3"><font size="2.7">&nbsp;&nbsp;&nbsp;4.3 Scene3</font></a></h3>
+				<h3 class="link"><a href="translation"><font size="2.7">&nbsp;&nbsp;&nbsp;4-5. Translation rate of protein</font></a></h3>
-				<h3 class="link"><a href="#fun4"><font size="2.7">&nbsp;&nbsp;&nbsp;4.4 Scene4</font></a></h3>
+				<h3 class="link"><a href="decomposition"><font size="2.7">&nbsp;&nbsp;&nbsp;4-6. Decomposition rate of O3C12HSL by AmiE</font></a></h3>
-				<h3 class="link"><a href="#modeling"><font size="2.7">&nbsp;&nbsp;&nbsp;4.5 modeling</font></a></h3>
+				<h3 class="link"><a href="degradation_amie"><font size="2.7">&nbsp;&nbsp;&nbsp;4-6. Degradation rate of AmiE</font></a></h3>
-				<h3 class="link"><a href="#HP">5．Integrated Human Practice</a></h3>
+				<h3 class="link"><a href="degradation_protein"><font size="2.7">&nbsp;&nbsp;&nbsp;4-7. Degradation rate of RFP and GFP</font></a></h3>
-				<h3 class="link"><a href="#SW"><font size="2.7">&nbsp;&nbsp;&nbsp;5.1 Snow White</font></a></h3>
+				<h3 class="link"><a href="#software">5. Software</a></h3>
-				<h3 class="link"><a href="#mirror"><font size="2.7">&nbsp;&nbsp;&nbsp;5.2 Mirror</font></a></h3>
+				<h3 class="link"><a href="#abstract"><font size="2.7">&nbsp;&nbsp;&nbsp;5-1. Abstract</font></a></h3>
-				<h3 class="link"><a href="#aca"><font size="2.7">&nbsp;&nbsp;&nbsp;5.3 ACA</font></a></h3>
+				<h3 class="link"><a href="#key_achievement"><font size="2.7">&nbsp;&nbsp;&nbsp;5-2. Key achievement</font></a></h3>
-				<h3 class="link"><a href="#reference">6. Reference</a></h3>
+				<h3 class="link"><a href="#work_flow"><font size="2.7">&nbsp;&nbsp;&nbsp;5-3. Work flow</font></a></h3>
+				<h3 class="link"><a href="#demo"><font size="2.7">&nbsp;&nbsp;&nbsp;5-4. Demonstration</font></a></h3>
+				<h3 class="link"><a href="#download"><font size="2.7">&nbsp;&nbsp;&nbsp;5-5. Download</font></a></h3>
 				</div><!-- /contents_menu -->
 			</div><!-- /contents_contents -->
 		</div><!-- /contents -->
+		<div id="overview" class="container">
+			<div id="overview_header" class="container_header">
+				<h2><span>1. Overview</span></h2>
+			</div><!-- /overview_header -->
+			<div id="overview_contents" class="container_contents">
+				<p class="normal_text">To reproduce the story of ”Snow White”, we have designed the cell-cell communication system with improved or characterized parts and collected data from comprehensive experiments. Furthermore, we constructed the mathematical model in order to simulate the behavior of the whole system and to confirm the feasibility of our story. This simulation successfully contributed to give the suggestions to wet lab experiments and confirm the feasibility of our system. In addition, in order to utilize TA (Toxin-Antitoxin) system, we developed a new software in Java for adjusting the number of ACA sequences, which MazF dimer recognizes and cleaves in mRNAs.</p>
+			</div><!-- /overview_contents -->
+		</div><!-- /overview -->
-		<div id="introduction" class="container">
+		<div id="story_simulation" class="container">
-			<div id="introduction_header" class="container_header">
+			<div id="story_simulation_header" class="container_header">
-				<h2><span>1. Introduction</span></h2>
+				<h2><span>2. Story Simulation</span></h2>
-			</div><!-- /_header -->
+			</div><!-- /story_simulation_header -->
-			<div id="introduction_contents" class="container_contents">
+			<div id="story_simulation_contents" class="container_contents">
-				<p class="normal_text">あなたがまだ幼かったころ、夜、眠る前のひと時、お母さん(ご両親？ばあば？)にせがんだことはありませんか？「お母さん、何か絵本読んで」と。白雪姫は世界で一番有名な童話といっても過言ではありません。(白雪姫は世界で一番有名な童話の一つです。) きっと一度は読み聞かせてもらったことでしょう。<br>
+			<div id="mathematical_model">
-				長く語り継がれてるお話にはたいてい教訓があります。白雪姫のお話から得られる教訓として主にあげられるのは次の二点だと思います。<br>
+				<div id="mathematical_model_header">
-				① 虚飾をはること、嫉妬することはよくないことである<br>
+					<h3><span>2-1. Mathematical Model</span></h3>
-				② むやみに知らない人を信用してはいけない<br>
+				</div><!-- /_header -->
-				単に優しいだけではいけません。美しい心とは、優しさや豊かさはもちろん、自らの気持ちをコントロールできる強い側面を持ち合わせている必要があります。<br>
+				<p class="normal_text">To simulate our gene circuits, we developed an ordinary differential equation model.</p>
-				私たちのプロジェクトでは、白雪姫の物語を外見の美しさと心の美しさという二つの美しさを総合的に評価していきます。
+				<p style="text-align:center;">[<a href="https://2016.igem.org/Team:Tokyo_Tech/Modeling_Details">Detailed Description for Modeling</a>]</p>
+				<p class="normal_text" style="text-align:center;"><a href="javascript:void(0);" onClick="show('modeling_detail');" class="showHidden">Read More</a></p>
+				<div id="modeling_detail" class="off">
+					<div id="modeling_detail_wrapper">
+						<div id="modeling_detail_expressions">
+							<h2>Differencial Equations</h2>
+							<h3>Snow White</h3>
+\begin{equation}
+\frac{d[mRNA_{RFP}]}{dt} = k - d[mRNA_{RFP}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{RFP}}})[mRNA_{RFP}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[mRNA_{RhlI}]}{dt} =  leak_{P_{lux}} + \frac{\kappa_{Lux}[C12]^{n_{Lux}}}{K_{mLux}^{n_{Lux}} + [C12]^{n_{Lux}}} - d[mRNA_{RhlI}] - F_{DiMazF}f[mRNA_{RhlI}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[RFP]}{dt} = \alpha [mRNA_{RFP}] - d_{RFP}[RFP]
+\end{equation}
+\begin{equation}
+\frac{d[RhlI]}{dt} = \alpha [mRNA_{RhlI}] - d_{RhlI}[RhlI]
+\end{equation}
+\begin{equation}
+\frac{d[C4]}{dt} = p_{C4}[RhlI]P_{Snow White} - d_{C4}[C4]
+\end{equation}
+\begin{equation}
+\frac{d[mRNA_{MazF}]}{dt} =  leak_{P_{lux}} + \frac{\kappa_{Lux}[C12]^{n_{Lux}}}{K_{mLux}^{n_{Lux}}+ [C12]^{n_{Lux}}} \\
+　　　　　　　- d[mRNA_{MazF}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazF}}})[mRNA_{MazF}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[mRNA_{MazE}]}{dt} = k - d[mRNA_{MazE}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazE}}})[mRNA_{MazE}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[MazF]}{dt} = \alpha [mRNA_{MazF}] - 2k_{Di_{MazF}}[MazF] + 2k_{-Di_{MazF}}[DiMazF] - d_{MazF}[MazF]
+\end{equation}
+\begin{equation}
+\frac{d[DiMazF]}{dt} = k_{Di_{MazF}}[MazF] - k_{-Di_{MazF}}[DiMazF] - 2k_{Hexa}[DiMazE][DiMazF]^2 \\
+　　　　　　　+ 2k_{-Hexa}[MazHexamer] - d_{DiMazF}[DiMazF] \end{equation}
+\begin{equation}
+\frac{d[MazE]}{dt} = \alpha [mRNA_{MazE}] - 2k_{Di_{MazE}}[MazE] + 2k_{-Di_{MazE}}[DiMazE] - d_{MazE}[MazE]
+\end{equation}
+\begin{equation}
+\frac{d[DiMazE]}{dt} = k_{Di_{MazE}}[MazE] - k_{-Di_{MazE}}[DiMazE] - k_{Hexa}[DiMazE][DiMazF]^2 \\
+　　　　　　　+ k_{-Hexa}[MazHexamer] - d_{DiMazE}[DiMazE]
+\end{equation}
+\begin{equation}
+\frac{d[MazHexa]}{dt} = k_{Hexa}[DiMazE][DiMazF]^2 - k_{-Hexa}[MazHexa] - d_{Hexa}[MazHexa]
+\end{equation}
+\begin{equation}
+\frac{dP_{Snow White}}{dt} = g \frac{E_{DiMazF}}{E_{DiMazF}+[DiMazF]}\left(1- \frac{P_{Snow White}+P_{Queen}+P_{Prince}}{P_{max}} \right) P_{Snow White}
+\end{equation}
+							<h3>Queen</h3>
+\begin{equation}
+\frac{d[mRNA_{GFP}]}{dt} = k - d[mRNA_{GFP}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{GFP}}})[mRNA_{GFP}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[mRNA_{LasI}]}{dt} =  leak_{P_{rhl}} + \frac{\kappa_{Rhl}[C4]^{n_{Rhl}}}{K_{mRhl}^{n_{Rhl}} + [C4]^{n_{Rhl}}} \\
+　　　　　　　- d[mRNA_{LasI}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{LasI}}})[mRNA_{LasI}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[GFP]}{dt} = \alpha [mRNA_{GFP}] - d_{GFP}[GFP]
+\end{equation}
+\begin{equation}
+\frac{d[LasI]}{dt} = \alpha [mRNA_{LasI}] - d_{LasI}[LasI]
+\end{equation}
+\begin{equation}
+\frac{d[C12]}{dt} = p_{C12}[LasI]P_{Queen} - d_{C12}[C12] - D[C12][AmiE]
+\end{equation}
+\begin{equation}
+\frac{d[mRNA_{MazF}]}{dt} =  leak_{P_{lux}} + \frac{\kappa_{Rhl}[C4]^{n_{Rhl}}}{K_{mRhl}^{n_{Rhl}} + [C4]^{n_{Rhl}}} \\
+　　　　　　　- d[mRNA_{MazF}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazF}}})[mRNA_{MazF}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[mRNA_{MazE}]}{dt} = k - d[mRNA_{MazE}] - F_{DiMazF}(1-(1-f)^{f_{mRNA_{MazE}}})[mRNA_{MazE}][DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[MazF]}{dt} = \alpha [mRNA_{MazF}] - 2k_{Di_{MazF}}[MazF] + 2k_{-Di_{MazF}}[DiMazF] - d_{MazF}[MazF]
+\end{equation}
+\begin{equation}
+\frac{d[DiMazF]}{dt} = k_{Di_{MazF}}[MazF] - k_{-Di_{MazF}}[DiMazF] - 2k_{Hexa}[DiMazE][DiMazF]^2 \\
+　　　　　　　+ 2k_{-Hexa}[MazHexamer] - d_{DiMazF}[DiMazF]
+\end{equation}
+\begin{equation}
+\frac{d[MazE]}{dt} = \alpha [mRNA_{MazE}] - 2k_{Di_{MazE}}[MazE] + 2k_{-Di_{MazE}}[DiMazE] - d_{MazE}[MazE]
+\end{equation}
+\begin{equation}
+\frac{d[DiMazE]}{dt} = k_{Di_{MazE}}[MazE] - k_{-Di_{MazE}}[DiMazE] - k_{Hexa}[DiMazE][DiMazF]^2 \\
+　　　　　　　+ k_{-Hexa}[MazHexamer] - d_{DiMazE}[DiMazE]
+\end{equation}
+\begin{equation}
+\frac{d[MazHexa]}{dt} = k_{Hexa}[DiMazE][DiMazF]^2 - k_{-Hexa}[MazHexa] - d_{Hexa}[MazHexa]
+\end{equation}
+\begin{equation}
+\frac{dP_{Queen}}{dt} = g \frac{E_{DiMazF}}{E_{DiMazF}+[DiMazF]}\left(1- \frac{P_{Snow White}+P_{Queen}+P_{Prince}}{P_{max}}\right) P_{Queen}\\
+\end{equation}
+							<h3>Prince</h3>
+\begin{equation}
+\frac{d[mRNA_{AmiE}]}{dt} = leak_{P_{lux}} + \frac{\kappa_{Lux}[C12]^n}{K_{mLux}^n + [C12]^n} - d[mRNA_{AmiE}]
+\end{equation}
+\begin{equation}
+\frac{d[AmiE]}{dt} = \alpha [mRNA_{AmiE}]P_{Prince} - d_{AmiE}[AmiE]
+\end{equation}
+\begin{equation}
+\frac{dP_{Prince}}{dt} = g\left(1- \frac{P_{Snow White}+P_{Queen}+P_{Prince}}{P_{max}}\right) P_{Prince}
+\end{equation}
+						</div><!-- /modeling_detail_expressions -->
+						<div id="modeling_detail_parameter">
+							<h2>Explanation about Parameters</h2>
+							<table border="1" style="margin: auto;">
+								<tbody>
+									<tr><td>Parameter </td><td> Description </td></tr>
+<tr><td>$$g$$ </td><td> Growth rate of each cells</td></tr>
+<tr><td>$$P_{max}$$ </td><td> Carrying capacity </td></tr>
+<tr><td>$$E_{DiMazF}$$ </td><td> Effect of MazF dimer on growth rate</td></tr>
+<tr><td>$$k$$ </td><td> Transcription rate of mRNA under \(P_{tet}\) </td></tr>
+<tr><td>$$leak_{P_{lux}}$$ </td><td> Leakage of \(P_{lux}\) </td></tr>
+<tr><td>$$leak_{P_{rhl}}$$ </td><td> Leakage of \(P_{rhl}\) </td></tr>
+<tr><td>$$\kappa_{Lux}$$ </td><td> Maximum transcription rate of mRNA under \(P_{lux}\)</td></tr>
+<tr><td>$$\kappa_{Rhl}$$ </td><td> Maximum transcription rate of mRNA under \(P_{rhl}\) </td></tr>
+<tr><td>$$n_{Lux}$$ </td><td> Hill coefficient for \(P_{lux}\)</td></tr>
+<tr><td>$$n_{Rhl}$$ </td><td> Hill coefficient for \(P_{rhl}\)</td></tr>
+<tr><td>$$K_{mLux}$$ </td><td> Lumped paremeter for the Lux System</td></tr>
+<tr><td>$$K_{mRhl}$$ </td><td> Lumped paremeter for the Rhl System</td></tr>
+<tr><td>$$F_{DiMazF}$$ </td><td> Cutting rate at ACA sequences on mRNA by MazF dimer</td></tr>
+<tr><td>$$f$$ </td><td> The probability of distinction of ACA sequencess in each mRNA</td></tr>
+<tr><td>$$f_{mRNA_{RFP}}$$ </td><td> The number of ACA sequences in \(mRNA_{RFP}\)</td></tr>
+<tr><td>$$f_{mRNA_{GFP}}$$ </td><td> The number of ACA sequences in \(mRNA_{GFP}\)</td></tr>
+<tr><td>$$f_{mRNA_{RhlI}}$$ </td><td> The number of ACA sequences in \(mRNA_{RhlI}\)　</td></tr>
+<tr><td>$$f_{mRNA_{LasI}}$$ </td><td> The number of ACA sequences in \(mRNA_{LasI}\)</td></tr>
+<tr><td>$$f_{mRNA_{MazF}}$$ </td><td> The number of ACA sequences in \(mRNA_{MazF}\)　</td></tr>
+<tr><td>$$f_{mRNA_{MazE}}$$ </td><td> The number of ACA sequences in \(mRNA_{MazE}\)　</td></tr>
+<tr><td>$$\alpha$$ </td><td> Translation rate of Protein </td></tr>
+<tr><td>$$k_{Di_{MazF}}$$ </td><td> Formation rate of MazF dimer </td></tr>
-				</p>
+<tr><td>$$k_{-Di_{MazF}}$$ </td><td> Dissociation rate of MazF dimer </td></tr>
+<tr><td>$$k_{Di_{MazE}}$$ </td><td> Formation rate of MazE dimer </td></tr>
+<tr><td>$$k_{-Di_{MazE}}$$ </td><td> Dissociation rate of MazE dimer </td></tr>
+<tr><td>$$k_{Hexa}$$ </td><td> Formation rate of Maz hexamer </td></tr>
+<tr><td>$$k_{-Hexa}$$ </td><td> Dissociation rate of Maz hexamer</td></tr>
+<tr><td>$$p_{C4}$$ </td><td> Production rate of C4HSL by RhlI</td></tr>
+<tr><td>$$p_{C12}$$ </td><td> Production rate of 3OC12HSL by LuxI </td></tr>
+<tr><td>$$D$$ </td><td> Decomposition rate of 3OC12HSL by AmiE </td></tr>
+<tr><td>$$d$$ </td><td> Degradation rate of mRNA </td></tr>
+<tr><td>$$d_{RFP}$$ </td><td> Degradation rate of RFP</td></tr>
+<tr><td>$$d_{GFP}$$ </td><td> Degradation rate of GFP </td></tr>
+<tr><td>$$d_{RhlI}$$ </td><td> Degradation rate of RhlI</td></tr>
+<tr><td>$$d_{LasI}$$ </td><td> Degradation rate of LasI</td></tr>
+<tr><td>$$d_{MazF}$$ </td><td> Degradation rate of MazF</td></tr>
+<tr><td>$$d_{DiMazF}$$ </td><td> Degradation rate of MazF dimer</td></tr>
+<tr><td>$$d_{MazE}$$ </td><td> Degradation rate of MazE </td></tr>
+<tr><td>$$d_{DiMazE}$$ </td><td> Degradation rate of MazE dimer </td></tr>
+<tr><td>$$d_{Hexa}$$ </td><td> Degradation rate of Maz Hexamer </td></tr>
+<tr><td>$$d_{C4}$$ </td><td> Degradation rate of C4HSL </td></tr>
+<tr><td>$$d_{C12}$$ </td><td> Degradation rate of 3OC12HSL </td></tr>
+<tr><td>$$d_{AmiE}$$ </td><td> Degradation rate of AmiE </td></tr>
-			</div><!-- /introduction_contents -->
+								</tbody>
-		</div><!-- /introduction -->
+							</table>
+						</div><!-- /modeling_detail_parameter -->
+					</div><!-- /modeling_detail_wrapper -->
+				</div><!-- /modeling_detail -->
+			</div><!-- /story_simalation_contents -->
-		<div id="story" class="container">
+<br>
-			<div id="story_header" class="container_header">
+			<div id="results">
-				<h2><span>2. Story</span></h2>
+				<div id="results_header">
-			</div><!-- /_header -->
+					<h3><span>2-2. Results</span></h3>
-			<div id="story_contents" class="container_contents">
+				</div><!-- /_header -->
-				<p class="normal_text"><span style="font-style : italic">Once upon a time, there lived a Queen.<br>
+　　 					<p class="normal_text">As a result, 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.
-				She was the fairest in the world and she herself also believed so.<br><br>
+                        </p>
+                <div align="center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tokyo_Tech--2-1-1.png" height ="300"><br></div>
-				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>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 5-1.Time-dependent change of the concentrations of fluorescence proteins</span>
-				“You are the fairest one of all.”<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>
-				“Magic Mirror on the wall, who is the fairest one of all?”<br><br>
-				One winter night, the Queen asked her mirror as usual.<br>
-				“Magic mirror on the wall, who is the fairest one of all?”<br>
-				The mirror answered,<br>
-				“Snow White is the fairest one.” <br><br>
-				The Snow White was Queen’s daughter-in-law.<br>
-				She was a kind and pure girl, and just turned seven years old.<br><br>
-				Then the Queen was shocked, and beside herself with rage. <br>
-				And thought,<br>
-				“If I kill Snow White, I would be the fairest one of all again.”<br><br>
-				The Queen prepared a poisoned apple.<br><br>
-				She decided to transform into a Witch and give the apple to Snow White.<br>
-				Snow White, who always takes people at their word , bit the apple, then sank into unconsciousness soon.<br><br>
-				The Dwarfs  found Snow White and they grieved her “death,” but they built a coffin and put her in it carefully.<br><br>
-				One day, a Price from a neighboring country passed by the Dwarfs’ house.<br><br>
-				Although he knew her death, he couldn’t help lifting her up because of her beauty.<br>
-				Then, she opened her eyes!<br><br>
-				This dislodged from Snow White’s throat the piece of poisoned apple that she had bitten off.<br><br><br>
-				Awakened Snow White was adored by everyone and lived happily ever after. <br><br><br>
-				THE END</span><br><br><br><br>
-				In our project, we will evaluate whether the story can be represented with the fluorescence intensity of RFP and GFP.<br>
-				The vertical axis of the graph shows the beauty of Snow White and the Queen, and the horizontal axis shows the elapsed time. In this project, the beauty is defined as follows.<br>
-				</p>
-				<p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Beauty = physical beauty + inner beauty</span>
-				</p><br>
-				<p class="normal_text">No matter how beautiful one’s physical appearance is, the one cannot be a real beauty unless has a beautiful mind. We will show you  which woman has real beauty, Snow White or the Queen.
-				</p>
-				<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tokyo_Tech--2-1-1.png" height ="300"><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>
 				</p></div><br>
+					<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/79/T--Tokyo_Tech--4koma.png" width ="800"class="align_right" /><br></div>
-				<p class="normal_text">  Even though we tried to evaluate the real beauty, unless the story is represented, we cannot do that. At first, we represented the story.<br>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 5-2.The story of "Snow White"</span>
+					<p class="normal_text">  In the blue area of Fig, the fluorescence intensity starts to increase. Snow White coli’s fluorescence intensity exceeds that of the Queen coli’s. It represents Snow White got fairer more and more. <br>
-				回路のデザインを紹介する前に、私たちのプロジェクトの根幹となるsystemを紹介します。<br>
+					In the pink area of Fig, C12 is being synthesized thanks to the C4’s appearance. And the concentration of C12 increases. As a result, the toxin MazF augments inside of Snow White coli and the Queen coli, suppressing the increment of fluorescent proteins. It is as if the Mirror’s answer transformed the Queen into a Witch, so she can give Snow White a poisoned apple.<br>
-				それはTA systemです。<br><br>
+					In the green area of Fig, C12 increases and the MazF inside Snow White coli induced by it increases even more. So the GFP exceeds the RFP. It looks as if Snow White bit the apple, sinking into unconsciousness promptly.<br>
+					In the yellow area of Fig, the AmiE synthesized by the introduced Prince coli decomposes the C12 molecules so the amount of MazF inside Snow White diminishes and the amount of C4 increases. It looks as if Prince lifted Snow White and she opened her eyes.<br>
-				TA systemとは、（以下略）
 				</p>
-			</div><!-- /story_contents -->
+				</div><!-- /results_contents -->
-		</div><!-- /story -->
+			</div><!-- /results -->
+			</div><!-- /story_simulation_contents -->
+		</div><!-- /story_smulation -->
+		<div id="fitting" class="container">
+			<div id="fitting_header" class="container_header">
+				<h2><span>3. Fitting</span></h2>
+			</div><!-- /fitting_header -->
+			<div id="fitting_contents" class="container_contents">
+				<p class="normal_text">TEST</p>
+			</div><!-- /fitting_contents -->
+		</div><!-- /fitting -->
-		<div id="design" class="container">
+		<div id="analysis" class="container">
-			<div id="design_header" class="container_header">
+			<div id="analysis_header" class="container_header">
-				<h2><span>3. Scene ＆ Design</span></h2>
+				<h2><span>4. Sensitivity Analysis</span></h2>
-			</div><!-- /_header -->
+			</div><!-- /analysis_header -->
-			<div id="design_contents" class="container_contents">
+			<div id="analysis_contents" class="container_contents">
-				<p class="normal_text">Our project begins from the scene that the Magic Mirror answers the question of the Queen.<br>
+				<p class="normal_text">We performed the sensitivity analysis descried in this section in order to examine which parameter dominates the story.</p>
-				We pick up 4 famous scenes and introduce them in order.　<br><br>
+<br>
-				<a href="#sce1">&nbsp;&nbsp;&nbsp;Scene1 : The Magic Mirror’s Answer</a><br>
+			<div id="requirements">
-				<a href="#sce2">&nbsp;&nbsp;&nbsp;Scene2 : The Queen’s Trap</a><br>
+				<div id="requirements_header">
-				<a href="#sce3">&nbsp;&nbsp;&nbsp;Scene3 : Snow White's Sleep</a><br>
+					<h3><span>4-1. Requirements</span></h3>
-				<a href="#sce4">&nbsp;&nbsp;&nbsp;Scene4 : The Prince’s Rescue</a><br>
+				</div><!-- /_header -->
-				</p><br><br>
+				<div id="requirements_contents">
-				<div id="sce1_header">
+					<p class="normal_text">We defined these requirements as the “successful Snow White story.”<br>
-					<h3><span>Scene1 : The Magic Mirror’s Answer</span></h3>
+)	At t = 150<br>
-				</div><!-- /_header -->
+					concentration of RFP > concentration of GFP<br>
-				<div id="sce1">
+)	At t = 700<br>
-				<div id="sce1_contents">
+					concentration  of RFP< concentration of GFP<br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f5/T--Tokyo_Tech--koma1.png" height ="200"><br></div>
+)	At t = 1500<br>
-					<p class="normal_text"><span style="font-style : italic">“Magic mirror on the wall, who is the fairest one of all?”<br>
+					concentration  of RFP > concentration of GFP<br>
-					The mirror answered,<br>
+					We the began to analyze the graph that satisfies these requirements that we could say that reproduces the story correctly. In the table bellow we show the range in which we modified each parameter. They were modified one step size at a time.<br></p>
-					“Snow White is the fairest one.” </span><br><br>
+					    <table border="1" style="margin: auto;">
+								<tbody>
-					The magic Mirror <span style="font-style: italic">coli</span> can produce RhlI protein under low temperature. Rhl protein production leads to production of the signaling molecule, C4HSL which tells the Queen that Snow White is the fairest, and the Queen <span style="font-style: italic">coli</span> receives this molecule.
+									<tr><td>Parameter </td><td> Range </td><td> Step size </td></tr>
+									<tr><td>$$D$$ </td><td> $$0.0001<D<0.001$$</td><td>$$0.0001$$</td></tr>
-					</p>
+									<tr><td>$$p_{C4}$$ </td><td> $$0.0001<p_{C4}<1$$</td><td>$$0.001$$</td></tr>
-				</div>
+									<tr><td>$$p_{C12}$$ </td><td> $$0.0001<p_{C12}<1$$</td><td>$$0.001$$</td></tr>
-				</div>
+									<tr><td>$$α$$ </td><td> $$0.01<α<0.2$$</td><td>$$0.01$$</td></tr>
+									<tr><td>$$d_{AmiE}$$ </td><td> $$0.001<d_{AmiE}<1$$</td><td>$$0.001$$</td></tr>
-				<div id="sce2_header">
+							    </tbody>
-					<h3><span>Scene2 : The Queen’s Trap</span></h3>
+					    </table>
-				</div><!-- /_header -->
+  <!--ここにレンジの表が入る-->
-				<div id="sce2">
-				<div id="sce2_contents">
+                   <p class="normal_text">As a result, we obtained the following parameter ranges.</p>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"><br></div>
+					    <table border="1" style="margin: auto;">
-					<p class="normal_text"><span style="font-style : italic">The Queen prepared a poisoned apple.<br>
+								<tbody>
-					She decided to transform into a Witch and give the apple to Snow White.</span><br><br>
+									<tr><td>Parameter </td><td> Value </td></tr>
+									<tr><td>$$D$$ </td><td> $$0.0056<D<0.001$$</td></tr>
-					 The Queen, which has received C4HSL, produces LasI and MazF.<br>
+									<tr><td>$$p_{C4}$$ </td><td> $$0.0029<p_{C4}<0.778$$</td></tr>
-					LasI produces signaling molecule, 3OC12HSL which represents the Poisoned Apple.<br>
+									<tr><td>$$p_{C12}$$ </td><td> $$0.001<p_{C12}<0.217$$</td></tr>
-					Additionally, produced MazF stops the translation in the Queen <span style="font-style : italic">coli</span>. MazE is incorporated  into the genetic circuit so that MazE expression does not stop translation immediately. If the translation stops 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.
+									<tr><td>$$α$$ </td><td> $$0.01<α<0.16$$</td></tr>
+									<tr><td>$$d_{AmiE}$$ </td><td> $$0.001<d_{AmiE}<1$$</td></tr>
-					</p>
+							    </tbody>
-				</div>
+					    </table>
-				</div>
+  <!--ここにレンジの表がもいっちょ入る-->
+</p>
-				<div id="sce3_header">
-					<h3><span>Scene3 : Snow White's Sleep</span></h3>
-				</div><!-- /_header -->
-				<div id="sce3">
-				<div id="sce3_contents">
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Tokyo_Tech--koma3.png" height ="200"><br></div>
-					<p class="normal_text"><span style="font-style : italic">Snow White, who always takes people at their word, bit the apple, then sank into unconsciousness soon.</span><br><br>
-					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> s.<br>
-					Snow White <span style="font-style : italic">coli</span>, which has received the C12HSL: the Poisoned Apple, expresses RhlI and MazF.<br>
-					Produced RhlI produces  a signaling molecule: C4HSL. As was described in the introduction of the Queen in the previous section , it can receive C4HSL and monitor Snow White <span style="font-style: italic">coli</span> through this.<br>
-					In addition, expressed MazF stops the translation in the Snow White <span style="font-style : italic">coli</span>.
-					</p>
-				</div>
-				</div>
-				<div id="sce4_header">
-					<h3><span>Scene4 : The Prince’s Rescue</span></h3>
-				</div><!-- /_header -->
-				<div id="sce4">
-				<div id="sce4_contents">
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png" height ="200"><br></div>
-					<p class="normal_text"><span style="font-style : italic">Although he knew her death, he couldn’t help lifting her up because of her beauty.<br>
-					Then, she opened her eyes!
-					</span><br><br>
-					 The Prince <span style="font-style: italic">coli</span> has received 3OC12HSL expresses AmiE. In general, it is said that AmiE degrades HSL with C8 or more. This leads to degradation of the Poisoned Apple that is 3OC12HSL.<br>
-					When the poison apple is degraded, MazF expression stops in Snow White, and gradually its function is canceled by MazE. Then, translation restarts in Snow White <span style="font-style: italic">coli</span>.
-					</p>
-				</div>
-				</div>
-			</div><!-- /design_contents -->
-		</div><!-- /design -->
-		<div id="function" class="container">
-			<div id="function_header" class="container_header">
-				<h2><span>4. Function</span></h2>
-			</div><!-- /_header -->
-			<div id="function_contents" class="container_contents">
-				<p class="normal_text">We conducted an experiment to confirm that the above four scenes can be represented. We introduced concepts in each scene and demonstrated them. Additionally, based on them, we simulated the representation of Snow White story.
-				</p>
-				<a href="#fun1">&nbsp;&nbsp;&nbsp;4.1. Scene1</a><br>
-				<a href="#fun2">&nbsp;&nbsp;&nbsp;4.2. Scene2</a><br>
-				<a href="#fun3">&nbsp;&nbsp;&nbsp;4.3. Scene3</a><br>
-				<a href="#fun4">&nbsp;&nbsp;&nbsp;4.4. Scene4</a><br>
-				<a href="#modeling">&nbsp;&nbsp;&nbsp;4.5. Modeling</a><br>
-				</p><br><br>
-				<div id="fun1_header">
-					<h3><span>4.1.　Scene1</span></h3>
-				</div><!-- /_header -->
-				<div id="fun1">
-				<div id="fun1_contents">
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f5/T--Tokyo_Tech--koma1.png" height ="200"><br></div>
-					<p class="normal_text">It is a cold inducible promoter (called Pcold commonly) that plays a most important role in this Scene. Unless the Magic Mirror answers the Queen’s question, this story never begins.
-					</p><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/97/T--Tokyo_Tech--assay_pcold.png" height ="200"><br></div>
-					<p class="normal_text">The experiment was conducted with BBa_1949001. We cultivated each sample at 18°C and 37°C and measured the GFP / Turbidity with a plate reader. The experimental result showed that samples cultured at 18°C had larger fluorescence intensity of GFP than those cultured at 37°C.<br>
-					Thus, we found that the story begins by lowering the culture temperature.
-				</div>
-				</div>
-				<div id="fun2_header">
-					<h3><span>4.2. Scene2</span></h3>
-				</div><!-- /_header -->
-				<div id="fun2">
-				<div id="fun2_contents">
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/8/8e/T--Tokyo_Tech--koma2.png" height ="200"><br></div>
-					<p class="normal_text">Snow White <span style="font-style: italic">coli</span> dies by the Poisoned Apple given by the Queen . The reporter with optimal strength is needed to work the genetic circuit which we designed properly. For this reason, we introduce rhl system assay as a concept in this Scene. We improved the function of rhl promoter through the process listed below and succeeded in newly getting  of rhl promoter mutants optimal for our project.<br><br>
-.2.1 reporter assay<br>
-					  First of all, we evaluated the activities the existing promoters: Prhl(BBa_I14017), Plux(BBa_R0062), Plas(BBa_R0079) when added 3 types of AHLs.
-					</p><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/00/T--Tokyo_Tech--assay_ahl_reporter.png" height ="200"><br></div>
-					<p class="normal_text">Although C12 is added to Plux, we found that the promoter activity can be seen. In addition, the graph shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when added C4.<br><br>
-.2.2 the simulation regarding the strength of rhl promoter<br>
+<br>
+			<div id="prince_coli">
+				<div id="prince_coli_header">
+					<h3><span>4-2. the Prince <span style ="font-style : italic">coli</span> should be put in in the middle</span></h3>
+				</div><!-- /_header -->
+				<div id="prince_coli_contents">
+					<p class="normal_text">We run simulations in order to determine whether we would get a better behavior let we introduce the Prince coli at the beginning or halfway of the story.</p>
+					<div align="left"><img src="https://static.igem.org/mediawiki/2016/1/1f/T--Tokyo_Tech--Population-miss.png" width ="500"class="align_left" /><br></div>
+				    <div align="left"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. ??.Number of individuals when the Prince <span style ="font-style : italic">coli</span> is introduced from the beginning</span>
+				    <div align="right"><img src="https://static.igem.org/mediawiki/2016/f/f1/T--Tokyo_Tech--AHL-miss.png" width ="500"class="align_right" /><br></div>
+				    <div align="right"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. ??.AHL concentrations when the Prince <span style ="font-style : italic">coli</span> is introduced from the beginning</span>
+                   <p class="normal_text">As a result, if we introduce the Prince coli from the beginning, the number of Prince coli increases too much (Fig) so the AmiE the Prince coli produces augments and the decomposition of C12 also overly occurs. So C12 is almost inexistent in the medium (Fig).</p>
+                   <div align="left"><img src="https://static.igem.org/mediawiki/2016/e/e7/T--Tokyo_Tech--Population.png" width ="500"class="align_left" /><br></div>
+				    <div align="left"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. ??.Number of individuals when the Prince <span style ="font-style : italic">coli</span> is introduced at t = 700</span>
+				    <div align="right"><img src="https://static.igem.org/mediawiki/2016/4/4f/T--Tokyo_Tech--AHL.png" width ="500"class="align_right" /><br></div>
+				    <div align="right"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. ??.AHL concentrations when the Prince <span style ="font-style : italic">coli</span> is introduced at t = 700</span>
+                   <p class="normal_text">On the other hand, if we introduce the Prince coli at t=700, the number of Prince coli does not increment much (Fig ), so C12 can exist until t=700 and then decreases thanks to the augment of AmiE (Fig ).<br>In conclusion, if we introduce the Prince coli at t=700, the circuit will behave accordingly.</p>
+</p>
-					Using the experimental results of preceding paragraph, we simulated to confirm that the genetic circuit we designed works properly when using the existing rhl promoter.
+<br>
-					</p><br>
+			<div id="prhl">
+				<div id="prhl_header">
+					<h3><span>4-3. Prhl should be changed</span></h3>
+				</div><!-- /_header -->
+				<div id="prhl_contents">
+					<p class="normal_text">In order to confirm whether by using a combination of the current promoters we would be able to reproduce the story with our gene circuit, we performed some simulations based on the results of our assays.</p>
 					<div align="center"><img src="https://static.igem.org/mediawiki/2016/6/68/T--Tokyo_Tech--projct_model1.png" height ="300"><br></div>
 					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> The intensity of Plux and Prhl promoters
-					</p></div><br>
+                   <p class="normal_text">The diagram above shows that the intensity of the two promoters should be in the red region of the figure.The combination of promoters we were originally going to use is shown in the graph by the green point. To move this point into the red region, we had to improve on Prhl so its expression level increases.</p>
+</p>
-					<p class="normal_text">The above diagram shows the intensity of the two promoters should be in the red region in the figure.<br>
+<br>
-					The above diagram shows the intensity of the two promoters should be in the red region in the figure. Because the green point shows the relationships of the promoters we intended to use primarily. In order to move to the red region, we found that it is needed to improve Prhl and increase its expression level.
+			<div id="production_ahl">
-					<br>
+				<div id="production_ahl_header">
+					<h3><span>4-4. Production rate of C4HSL and 3OC12HSL by RhlI and LasI</span></h3>
+				</div><!-- /_header -->
+				<div id="production_ahl_contents">
+					<p class="normal_text">The signaling molecule production rates by RhlI and LasI can be changed by modifying RhlI and LasI to make more or less signaling molecule in silico.</p>
+					<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/9f/T--Tokyo_Tech--Production-AHL.png" height ="300"><br></div>
+					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> Concentrations of GFP and RFP dependencies of production rate of C4 by RhlI
+                   <p class="normal_text">Each line corresponds to the transition of the concentration of RFP/GFP with a certain production rate of C12. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher production rate of C12.<br>
+                   if the production rate of C4 is between 0.029 and 0.778, our system can reproduce the story.<br>
+                   If this parameter is too small, The production of LasI by the Queen <span style ="font-style : italic">coli</span> is insufficiently inhibited by MazF so C12 increases greatly. As a result, The concentration of GFP overcomes the concentration of RFP and the story does not develop correctly. And if this parameter is too big, The production of LasI by the Queen <span style ="font-style : italic">coli</span>  is overly inhibited by MazF so C12 does not increase. As a result, the concentration of RFP is always greater than the GFP concentration and the story does not develop either.</p>
+                   <div align="center"><img src="https://static.igem.org/mediawiki/2016/0/05/T--Tokyo_Tech--Production-AHL2.png" height ="300"><br></div>
+					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> Concentrations of GFP and RFP dependencies of production rate of C12 by LasI
+                   <p class="normal_text">Each line corresponds to the transition of the concentration of RFP/GFP with a certain production rate of C4. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher production rate of C4.<br>
+                    If production rate of C12 is between 0.001 and 0.217, our system can reproduce the story.</p>
+</p>
-.2.3　Rhl system Assay<br>
+<br>
-					We made mutants of rhl promoter by inserting a point mutation into the wild type (WT) rhl promoter.<br>
+			<div id="translation">
-					In the experiment, we added reagent AHLs into the reporters and examined their fluorescence intensities.<br>
+				<div id="translation_header">
-					As a result, we succeeded in newly obtaining stronger mutants than the WT, Prhl (M) and Prhl the (NM).
+					<h3><span>4-5. Translation rate of protein</span></h3>
-					</p><br>
+				</div><!-- /_header -->
+				<div id="translation_contents">
+					<p class="normal_text">Translation rate affects the production of protein.</p>
+					<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/03/T-Tokyo_Tech--Translation.png" height ="300"><br></div>
+					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> Concentrations of GFP and RFP dependencies of translation rate of proteins
+                   <p class="normal_text">Each line corresponds to the transition of the concentration of RFP/GFP with a certain translation rate. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher translation rate.<br>
+                   If translation rate of protein is between 0.01 and 0.16, our system can reproduce the story.
+</p>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/c/c6/T--Tokyo_Tech--assay_prhl1.png" height ="300"><br></div>
+<br>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span>
+			<div id="decomposition">
-					</p></div><br>
+				<div id="decomposition_header">
+					<h3><span>4-6. Decomposition rate of C12 by AmiE</span></h3>
+				</div><!-- /_header -->
+				<div id="decomposition_contents">
+					<p class="normal_text">AmiE decomposes C12. The concentration of C12 after input of the Prince <span style ="font-style : italic">coli</span> is changed by AmiE. If the decomposition rate of C12 is too small, C12 does not decrease enough so the MazF inside Snow White <span style ="font-style : italic">coli</span> continues being expressed and the concentration of RFP decreases.</p>
+					<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/08/T--Tokyo_Tech--Decomposition.png" height ="300"><br></div>
+					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> Concentrations of GFP and RFP dependencies of decomposition rate of C12 by AmiE
+                   <p class="normal_text">Each line corresponds to the transition of the concentration of RFP/GFP with a certain value of D. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher value of decomposition rate of C12.<br>
+                   If degradation rate of C12 is higher than 0.00056, our system can reproduce the story.</p>
+</p>
-					<p class="normal_text"> iGEM Tokyo_Tech has Improved rhl promoter in the past. However, comparing the Prhl (NM) to Prhl (LR), the SN ratio of Prhl (NM) was higher than that of Prhl (LR) . Futuremore, the result shows that Prhl (LR)has crosstalk with C12 at a constant rate. When representing Snow White story, and the crosstalk to C12 happens, the Queen <span style="font-style : italic">coli</span> will suicide by eating the Poisoned Apple which she has made by herself. Therefore, we made the mutants, using WT as a template.</p>
+<br>
+			<div id="degradation _amie">
+				<div id="degradation_amie_header">
+					<h3><span>4-7. Degradation rate of AmiE</span></h3>
+				</div><!-- /_header -->
+				<div id="degradation_amie_contents">
+				<div align="center"><img src="https://static.igem.org/mediawiki/2016/d/d4/T--Tokyo_Tech--DegradationAmiE.png" height ="300"><br></div>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> Concentration of GFP and RFP dependencies of degradation rate of AmiE
+                 <p class="normal_text">Each line corresponds to the transition of the concentration of RFP/GFP with a certain degradation rate of AmiE. Red and green lines correspond to RFP and GFP, respectively. Blighter one indicates higher degradation rate of AmiE.<br>
+                   Even if we modify the values of the parameters inside the defined range, the concentration of RFP overcomes the concentration of GFP. And even if the degradation rate of AmiE is small, the decomposition rate of C12 by AmiE is high enough so C12 decreases sufficiently.</p>
+</p>
+<br>
+			<div id="degradation_protein">
+				<div id="degradation_protein">
+					<h3><span>4-8. Degradation rate of RFP and GFP</span></h3>
+				</div><!-- /_header -->
+				<div id="degradation_protein_contents">
+					<p class="normal_text">The degradation rate of RFP and GFP is key to the success the story of ‘Snow White’.<br>
+					These parameters are closely related to the concentrations of GFP and RFP, so we conjectured that if they do not take appropriate values the story can not be correctly reproduced.</p>
+					<div align="center"><img src="https://static.igem.org/mediawiki/2016/a/a0/T--Tokyo_Tech--DegradationGFP.png" height ="300"><br></div>
+					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> Relation of degradation rate of GFP and RFP
+                   <p class="normal_text">The story is reproduced only if the degradation rate of RFP and GFP are the same.</p>
+</p>
+		</div><!-- /analysis_contents -->
+		</div><!-- /analysis -->
+<br>
+		<div id="software" class="container">
+			<div id="software_header" class="container_header">
+				<h2><span>5. Software</span></h2>
+			</div><!-- /software_header -->
+			<div id="software_contents" class="container_contents">
+<br>
+			<div id="abstract">
+				<div id="abstract_header">
+					<h3><span>5-1. Abstract</span></h3>
+				</div><!-- /_header -->
+				<div id="abstract_contents">
+				<img src="https://static.igem.org/mediawiki/2016/8/8b/T--Tokyo_Tech--ACA_Dwarfs.jpg" height ="300"><br></div>
+					<p class="normal_text">We developed a new software named ACADwarfs. This software helps to control the sensitivity of the protein to MazF by regulating the number of ACA sequences in the mRNA sequence. ACADwarfs can increase or decrease the number of ACA sequences on mRNA without changing the amino acid sequences that the mRNA specifies or frameshifts resulted from insertion of bases without considering. <br>Therewith we improve the practicality of the characteristic of the <span style ="font-style : italic"> mazEF </span> system. For example you can let protein A express constantly by eliminating ACA sequences of the sequence, while letting protein B suspend to express, at the desirable timing, by expression of MazF.<br>This software also evades using rare codons, so you don’t have to worry about them.</p>
+<br>
+			<div id="key_achievement">
+				<div id="key_achievement_header">
+					<h3><span>5-2. Key achievement</span></h3>
+				</div><!-- /_header -->
+					<p class="normal_text">
+・Provided the tool regulating the number of ACA sequences<br>
+・Released under open-source license so everyone can use it<br>
+・Able to correspond to any base arrangements<br>
+・Rare codons are evaded<br>
+・Extend the application field of <span style ="font-style : italic">mazE</span> system<br>
+</p>
+<br>
-				</div>
+			<div id="work_flow">
-				</div>
+				<div id="work_flow_header">
+					<h3><span>5-3. Work flow</span></h3>
-				<div id="fun3_header">
+				</div><!-- /_header -->
-					<h3><span>4.3. Scene3</span></h3>
+					<p class="normal_text">TEST
+</p>
+<br>
+			<div id="demo">
+				<div id="demo_header">
+					<h3><span>5-4. Demonstration</span></h3>
+				</div><!-- /_header -->
+					<p class="normal_text">TEST
+</p>
+<br>
+			<div id="download">
+				<div id="download_header">
+					<h3><span>5-5. Download</span></h3>
 				</div><!-- /_header -->
-				<div id="fun3">
+				<p class="normal_text">To download click <a href="https://static.igem.org/mediawiki/2016/3/32/T--Tokyo_Tech--ACA_Dwarfs.zip">here</a>.</p>
-				<div id="fun3_contents">
+				<p class="normal_text">The code is available on <a href="https://github.com/Ryuta339/ACADwarfs">github</a>.</p>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/6/6f/T--Tokyo_Tech--koma3.png" height ="200"><br></div>
+			</div><!-- /software_contents -->
-					<p class="normal_text">In this Scene, we introduce TA system as a concept.<br>
+		</div><!-- /software -->
-					If we do not show  that translation is stopped by MazF, and translation is restarted by MazE, we cannot represent the key part of the story that "Snow White falls down by the Poisoned Apple and wake up again by the Prince.”
+	</div><!-- /main_contents -->
-					</p><br>
+	<script type="text/javascript">
+		function show(idName){
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/42/T--Tokyo_Tech--maz1.png" height ="200"><br></div>
+			var elem = document.getElementById(idName);
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span>
+			if(elem.className === 'on'){
-					</p></div><br>
+				elem.className = 'off';
+				var elem2 = document.getElementsByClassName('showHidden');
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tokyo_Tech--maz2.png" height ="200"><br></div>
+				elem2[0].textContent = 'Read More';
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span>
+			} else {
-					</p></div><br>
+				elem.className = 'on';
+				var elem2 = document.getElementsByClassName('showHidden');
-					<p class="normal_text">In this experiment, we used the BBa_K1949100 and Bba_1949102.<br>
+				elem2[0].textContent = 'Hide';
-					First, MazF was expressed by arabinose, and 2 h later, the MazE was expressed by IPTG.<br>
+			}
-					From the experimental results, we found that the turbidity of samples without MazE turbility did not rise. However, we also found that <span style="font-style: italic">E. coli</span> restarts its cell growth when MazE is expressed by adding IPTG.<br>
+		}
-					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>
+	</script>
-					From the above, we concluded 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.<br>
-					According to the experiments, we showed that TA system works properly.
-					</p>
-				</div>
-				</div>
-				<div id="fun4_header">
-					<h3><span>4.4. Scene4</span></h3>
-				</div><!-- /_header -->
-				<div id="fun4">
-				<div id="fun4_contents">
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/40/T--Tokyo_Tech--koma4.png" height ="200"><br></div>
-					<p class="normal_text">In the final Scene, we introduce the selective degradation of AHLs by AmiE as a concept.<br>
-					As shown in the TA system, we found that Snow White can wake up again because MazE counteracts the function of MazF. However, we have not showed the trigger, that is, the degradation of 3OC12HSL by the Prince coli. Therefore, we examined whether AmiE selectively degrades AHLs.<br>
-					</p><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/75/T--Tokyo_Tech--6-pr-4-1.png" height ="250"><img src="https://static.igem.org/mediawiki/2016/9/94/T--Tokyo_Tech--6-pr-4-2.png" height ="250"></div><br>
-					<p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> AmiE degrades C12  &nbsp;
-					<span style="font-weight: bold;">Fig.  </span> AmiE barely degrades C４ <br>
-					C12 is greatly degraded. On the other hand, the C4 is barely degradedPrhl(LR).
-					</p><br>
-					<p class="normal_text">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.<br>
-					For these reasons, we showed that AmiE selectively degrades AHLs, only C12 in this project.
-					</p>
-				</div>
-				</div>
-				<div id="modeling_header">
-					<h3><span>4.5. Modeling</span></h3>
-				</div><!-- /_header -->
-				<div id="fun4">
-				<div id="fun4_contents">
-					<p class="normal_text">From the results of Wet , it was showed that the designed circuit works properly at each point. We simulated the representation of the story in a combination of these Points.
-					<br><br>
-.5.1　王子様いつやってくのだろうか<br>
-					We simulated to confirm which is better, adding the Prince <span style="font-style: italic">coli</span> first or adding it in midstream, to make our genetic circuit work. <br>
-					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 degraded too much. Then, C12 cannot exist in the medium (Fig. 2) and the circuit does not work correctly.
-					</p><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/1/1a/T--Tokyo_Tech--project_model1.png" height ="300"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span>王子を初めから入れた場合の個体数
-					</p></div><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/5/59/T--Tokyo_Tech--project_model2.png" height ="300"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span>王子を初めから入れた場合のAHL
-					</p></div><br>
-					<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).
-					</p><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/1/1f/T--Tokyo_Tech--project_model3.png" height ="300"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span>王子をt = 700で投入した場合の個体数
-					</p></div><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/c/cc/T--Tokyo_Tech--project_model4.png" height ="300"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span>王子をt = 700で投入した場合のAHL
-					</p></div><br>
-					<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> when t equals 700.<br><br>
-.5.2　ストーリーの再現<br>
-					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.
-					</p><br>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tokyo_Tech--2-1-1.png" height ="300"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. </span>time-dependent change of the concentrations of the two fluorescence proteins
-					</p></div><br>
-					<p class="normal_text">シミュレーションをもとに、白雪姫と女王様のどちらが真に美しいのか見てみましょう。 </p>
-					<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/79/T--Tokyo_Tech--4koma.png" height ="500"><br></div>
-					<p class="normal_text">In the blue area of Fig, ここでは、白雪姫の蛍光強度が、女王様の蛍光強度を上回っています。<br>
-					ある日を境に、白雪姫の美しさが女王の美しさを超えたからです。It represents Snow White got fairer more and more.<br>
-					In the pink area of Fig, 投入されたC4によりC12が生産さます。これによりトキシンであるMazFが継母の個体内で増加し、蛍光タンパク質の増加を抑制します。It looks as if after mirror’s answer Queen transformed into a Witch and gave the apple to Snow White.これは女王様が嫉妬に怒り狂い「犯罪」に手を染めてしまった場面にあたります。<br>
-					In the green area of Fig, C12が増加してC12によって誘導される白雪姫内のMazFがより増加し、GFPがRFPを上回ります。It looks as if Snow White bit the apple, then sank into unconsciousness soon<br>
-					In the yellow area of Fig, 投入された王子が生産したAmiEによりC12が分解されるため白雪姫のMazFが減少してC4が増加します。It looks as if Prince lifted Snow White and she opened her eyes. <br>
-					以上より、私たちがデザインした回路はストーリーを再現でき、また、真に美しいのは白雪姫であるということがわかりました。
-					</p>
-				</div>
-				</div>
-			</div><!-- /function_contents -->
-		</div><!-- /function -->
-		<div id="HP" class="container">
-			<div id="HP_header" class="container_header">
-				<h2><span>5．Integrated Human Practice</span></h2>
-			</div><!-- /_header -->
-			<div id="HP_contents" class="container_contents">
-				<p class="normal_text">5.1 Snow White<br>
-.2 Mirror<br>
-.3 ACA<br><br><br>
-.1 Snow White<br>
-  				Why we are going to participate in the iGEM with the theme of Snow White? It is resulted from drawing on the opinion obtained through various dialogues with the public. This time, we decided to deal with Snow White, which was easy-to-follow, familiar, and attracted to the public.<br><br>
-.2 Addition of other characters<br>
-  				Initially, only 3 characters, Snow White, the Queen, and the Prince, appeared in our story. However, we got the simple question from junior high school and high school students that why other characters did not appear in our story, we decided to add others. We designed the Magic Mirror’s genetic circuit and conducted an experiment. Additionally, as dwarfs, we prepared the <span style="font-style: italic">E. coli</span> (strain name). As you can see in the photograph, they have round shapes compared to the general <span style="font-style: italic">E. coli</span> and are very charming. <br>
-  				</p>
-  				<div align="center"><img src="url" height ="200"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. </span>the Dwarf <span style="font-style : italic">coli</span>
-					</p></div><br>
-				<div align="center"><img src="https://static.igem.org/mediawiki/2016/d/df/T--Tokyo_Tech--ACADwarfs.png" height ="200"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. </span>ACA Dwarfs
-					</p></div><br>
-				<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 the insertion of bases without considering.
-				</p><br>
-				<div align="center"><img src="https://static.igem.org/mediawiki/2016/c/c9/T--Tokyo_Tech--ACAmodel1.jpg" height ="200"><br></div>
-					<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. </span>
-					</p></div><br>
-				<p class="normal_text">  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.
-				</p>
-			</div><!-- /HP_contents -->
-		</div><!-- /HP -->
-		<!-- <div id="reference" class="container">
-			<div id="reference_header" class="container_header">
-				<h2><span>6. Reference</span></h2>
-			</div><!-- /_header -->
-			<!--<div id="reference_contents" class="container_contents">
-				<p class="normal_text">nakamidayo
-				</p>
-			</div><!-- /reference_contents -->
-		<!-- </div><!-- /reference -->
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Difference between revisions of "Team:Tokyo Tech/Project"

Revision as of 06:15, 18 October 2016

Model

Contents

1. Overview

2. Story Simulation

2-1. Mathematical Model

Differencial Equations

Snow White

Queen

Prince

Explanation about Parameters

2-2. Results

3. Fitting

4. Sensitivity Analysis

4-1. Requirements

4-2. the Prince coli should be put in in the middle

4-3. Prhl should be changed

4-4. Production rate of C4HSL and 3OC12HSL by RhlI and LasI

4-5. Translation rate of protein

4-6. Decomposition rate of C12 by AmiE

4-7. Degradation rate of AmiE

4-8. Degradation rate of RFP and GFP

5. Software

5-1. Abstract

5-2. Key achievement

5-3. Work flow

5-4. Demonstration

5-5. Download