Revision as of 05:46, 19 October 2016

3-1-2 mazEF system Assay

3-1-2-1 Stop & Go

1-1. Introduction

The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, mazE expression was induced by the addition of IPTG(2 mM) after mazF expression was induced by the addition of arabinose(0.02%). As a result, it was able to resuscitate from a state of being inhibited cell growth. We named this experiment as "Stop & Go" because it was to resuscitate growth from inhibiting cell growth.

1-2. Summary of the Experiment

Transformants as shown below was prepared.

E. coli A : carrying the Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli A

E. coli C : carrying the PBAD-rbs (pSB6A1) , Plac-rbs (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli C

E. coli B : carrying the PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli B

E. coli D : carrying the PBAD-rbs -mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs-mazE (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli D

It has been found that the expression of Plac is larger than that of PBAD. Therefore, we expected that the expression of mazE would be finally larger than the that of mazF. Samples were incubated with vigorous shaking at 37℃. When turbidity reached 0.03, 0.02% arabinose was added to induce mazF expression. Two hours after the addition of arabinose, 2 mM IPTG was added to induce mazE expression. The turbidity and Relative Fluorescece Units (RFU) of GFP were measured at several time points.

1-3. Results

From the result in Fig. 3-1-2-1-3-1, it was found that MazF inhibited cell growth. The mazE expression was induced 2 h after the mazF expression, and about 8 h later, cell growth resumed. Similarly, the RFU of GFP was also resumed(Fig. 3-1-2-1-3-2).

Fig. 3-1-2-1-3-1 time vs Turbidity (Stop & Go)

Fig. 3-1-2-1-3-2 time vs RFU of GFP (Stop & Go)

4. Discussion

　Before starting the experiments, we anticipated that recovery from the growth inhibition by MazF would be observed immediately after the induction of mazE expression. However, it took 8 h for us to observe the recovery.

5. Materials and Methods

5-1. Construction

-Strain All the samples were XL1-Blue strain.
-Plasmids
E. coli A : Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)

E. coli C : PBAD-rbs (pSB6A1) , Plac-rbs (pSB3K3)

E. coli B : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)

E. coli D : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs-mazE (pSB3K3)

PBAD (BBa_I0050) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) , rbs (BBa_B0034) , mazF (BBa_K1096002) , tt (BBa_B0015)

5-2. Assay Protocol

Pre-culture
1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).

2. Incubate with vigorous shaking for 12 h.

　　 Incubation and Assay
1. Measure the turbidity of the pre-cultures.

2. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.

3. Incubate with vigorous shaking so that turbidity becomes 0.03.

4. Add arabinose so that the final concentration becomes 0.02%.

5. 2 h after the addition of arabinose, we added IPTG so that the final concentration becomes 2 mM.

6. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at the proper time.

3-1-2-1 Go & Stop

2-1. Introduction

In Experiment 1-2-1, we found that a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what will happen when a toxin is expressed after the antitoxin constitutive expression? Therefore, we conducted the experiment. Since cell growth was resuscitated after cells had grown, we named this experiment, "Go & Stop".

2-2. Summary of the Experiment

Transformants as shown below was prepared. E. coli A : carrying the Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli A

E. coli C : carrying the PBAD-rbs (pSB6A1) , Plac-rbs (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli C

E. coli E : carrying the PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , vector (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli E

E. coli F : carrying the PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs-mazE (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli F

E. coli G : carrying the PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs(weak)-mazE (pSB3K3)

Fig. 3-1-2-2-1 Plasmid diagram of E. coli G

In order to adjust the amount of expression of mazE, two types of RBS (B0034 and J61117: J61117 is weaker than B0034) was used. Using these plasmids, we tried clarifying stoichiometric relation between A and B by changing the expression of MazE. Samples were incubated with vigorous shaking at 37℃. When turbidity reached 0.03, 0.02% arabinose was added to induce mazF expression. The turbidity and Relative Fluorescece Units (RFU) of GFP were measured at several time points.

2-3. Results

Increase in turbidity and RFU of E. coli G stopped earlier than that of E. coli F(Fig. 3-1-2-2-3-1 and Fig. 3-1-2-2-3-2). As a result, turbidity and the RFU of E. coli F in the stationary phase was smaller than that of E. coli G.

Fig. 3-1-2-2-3-1.Time vs Turbidity (Go &Stop)

Fig. 3-1-2-2-3-2Time vs RFU of GFP (Go &Stop)

Calculation of the change of RFU of GFP / Turbidity per unit time (translation efficiency) indicates that the expression level of MazE correlated with the translation efficiency(Fig. 3-1-2-2-3-3).

Fig. 3-1-2-2-3-3translation efficiency of each E. coli.

2-4. Discussion

We found that MazF inhibited cell growth and translation even when there was MazE. In addition,these results suggested that there was a relation between the resuscitation and mazE expression.

From results of Experiment1.2.1. and Experiment1.2.2., it was expected that mazEF can repeatedly control cell growth.

2-5. Materials and Methods

2-5-1. Construction

-Strain
All the samples were XL1-Blue strain.

-plasmid
E. coli C : PBAD-rbs (pSB6A1) , Plac-rbs (pSB3K3)

E. coli A : Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)

E.coli E : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , vector (pSB3K3)

E. coli G : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs(BBa_J61117)-mazE (pSB3K3)

E. coli F : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs(BBa_B0034)-mazE (pSB3K3)

PBAD (BBa_I0050) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) , rbs (BBa_B0034) , mazF (BBa_K1096002) , mazF (BBa_K1096001),tt (BBa_B0015) , weak rbs (BBa_J61117)

2-5-2. Assay Protocol

Pre-culture
1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).
2. Incubate with vigorous shaking for 12 h.

Incubation and Assay
1. Measure the turbidity of the pre-cultures.
2. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.
3. Incubate with vigorous shaking so that turbidity becomes 0.03
4. Add arabinose so that the final concentration becomes 0.02%.
5. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at proper times.

3-1-2-3 mazEF System Assay on the LB Agar Plate

3-1. Introduction

　The control of cell growth by the mazEF system has been shown until the previous sections. In this section, we analyzed whether the “stop & go” experiment can be repeated many times.

2-2. Summary of the Experiment

A pSB6A1-based plasmid containing both the PBAD (BBa_I0050) - rbs (BBa_B0034) - mazF (BBa_K1096002) cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac (BBa_R0010) - rbs (BBa_B0034) - mazE (BBa_K1096001) cassette was constructed. These plasmids were co-introduced into E. coli. We prepared LB media containing arabinose (Ara(+)), IPTG (IPTG (+)), both inducers (Ara(+), IPTG(+)), and no inducers (Ara (-), IPTG (-)). We tested whether the E. coli cells formed colonies on above plates by controlling the mazEF system. The procedures are shown below.

(i) Day1 : Each transformant was streaked onto the Ara(+)-, IPTG(+)-, and
Ara(-)_IPTG(-)-plates.

(ii) Day2 : The colonies on the above plates were re-streaked onto the Ara(+)_IPTG(+)-plate. Even when no colonies were found on the plates, the streaked areas on day 1 were
scratched by toothpicks and re-streaked.

(iii) Day3 : The same procedure was conducted as day 2 except that Ara(+)-plate was used.

(iv) Day4 : The same procedure was conducted as day 2.

Fig. 3-2-1-3-1. Overview of the experiment

3-3. Results & Discussion

From the result, it was clarified that growth of E. coli cells was repeatedly controlled by expression of mazE(Fig. 3-1-2-3-3-1 and Table 3-1-2-3-3-1). The results of this experiment are very useful in our project, and it is expected to lead to new biotechnological applications.

Fig. 3-1-2-3-3-1 Results of culture that was controlled TA system

Table 3-1-2-3-3-1 Results of culture that was controlled TA system

From the above result, it was clarified that growth of E. coli cells was repeatedly controlled by expression of mazE. The results of this experiment are very useful in our project, and it is expected to lead to new biotechnological applications.

3-5. Materials and Methods

3-5-1. Construction

-Strain

All the samples were XL1-Blue strain.

-Plasmids

E. coli D: PBAD-rbs-mazF (pSB6A1)　，　Plac-rbs-mazE (pSB3K3)

E. coli H : PBAD-rbs-mazF(pSB6A1)　，　Plac-rbs (pSB3K3)

E. coli I : PBAD-rbs (pSB6A1)　，Plac-rbs (pSB3K3)

PBAD : BBa_I0050 , Plac : BBa_R0010 , rbs : BBa_B0034 , mazF : BBa_K1096002 , mazE : BBa_K1096001

3-5-2. Assay Protocol

1) Making LB agar medium containing arabinose and IPTG.

2) E. coli are applied at 3 agar medium (in arabinose, in IPTG, in arabinose and IPTG)

3) Overnight culture at 37°C for 24 h

4) To confirm TA system, inoculate colonies of E. coli having plasmidⅠ, Ⅱ, Ⅴ at agar medium
containig arabinose and IPTG

5) Overnight culture at 37°C for 24 h

6) Inoculate colonies of E. coli into agar medium containing arabinose.

7) Overnight culture at 37°C for 24 h

8) Inoculate colonies of E. coli into agar medium in arabinose and IPTG

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-			<h1 align="center">Human Practices</h1>
+			<h1 align="center">3-1-2 <span style="font-style : italic">mazEF</span> system Assay</h1>
 		</div><!-- /page_header -->
 		<div id="contents" class="container">
 			<div id="contents_header" class="container_header">
-				<h1><span>Contents</span></h1>
+				<h2><span>Contents</span></h2>
 			</div><!-- /contents_header -->
 			<div id="contents_contents" class="container_contents">
 				<div id="contents_menu">
-				<h3 class="link"><a href="#introduction">1. Overview</a></h3>
+				<h3 class="link"><a href="#SaG"><font size="5">1. Stop & Go</font></a></h3>
-				<h3 class="link"><a href="#overview">2. 3E</a></h3>
-				<h3 class="link"><a href="#3E">3. Policy and Practices</a></h3>
-				<h3 class="link"><a href="#Policy_and_Practices">4. Snow White versions</a></h3>
+				<h3 class="link"><a href="#GaS"><font size="5">2. Go & Stop</font></a></h3>
+				<h3 class="link"><a href="#Queen"><font size="5">3. <span style ="font-style : italic">mazEF</span> System Assay on the LB Agar Plate</font></a></h3>
 				</div><!-- /contents_menu -->
 			</div><!-- /contents_contents -->
 		</div><!-- /contents -->
-		<div id="overview" class="container">
-			<div id="overview_header" class="container_header">
-				<h1><span>1. Overview</span></h1>
-			</div><!-- /overview_header -->
-			<div id="overview_contents" class="container_contents">
-				<p class="normal_text">At the beginning of our iGEM activities, we felt that people seem to be unfamiliar with the synthetic biology and its social contribution. In order to solve these issues, we chose to show our concepts through Snow White, one of the most famous stories around the world. Besides, we hold many outreach activities,   dialogued with experts, and created a model called “3E”: education, ethics, and economy. We also designed some ideas base to our research (Toxin-Antitoxin system etc.) for society. We improved our project by feedback of these human practice activities. <br>
-We could improve the public conscious to synthetic biology and our project mutually by communicating with people.
-                                 </p>
-			</div><!-- /overview_contents -->
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-		<div class="flexslider">
-			<ul class="slides">
-			</ul>
-		</div>
-		<div id="3E" class="container">
+		<div id="SaG" class="container container_top2">
-			<div id="3E_header" class="container_header">
+			<h1 align="center">3-1-2-1 Stop & Go</h1>
-				<h1><span>2. 3E</span></h1>
+		</div><!-- /page_header -->
-			</div><!-- /3E_header -->
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+				<h2><span>Contents</span></h2>
-				<span id="ethics_string" class="string_image"></span>
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-				<p class="normal_text">We take our project into consideration from three aspects: education, ethics, and economy. From these three aspects, we can approach in various ways, thereby we can grasp our project multilaterally. So far, we had worked on many activities to inform the public distinctly of <span style="font-style: italic">E. coli</span>, genetic modification, and synthetic biology. Once we faced some issues, we had many discussions and tried solving the problems. Secondly, we had thought about ethics as a key point in our project and made ethics code of iGEM 2016 Tokyo_Tech Team. Furthermore, we coordinated activities to achieve the protein production control using TA systems. Although it is difficult to use commercially at present, it is a great achievement that we created the idea and technology regarded as a steppingstone. This page shows all of our human practice activities.
-				</p>
-			</div><!-- /3E_contents -->
-			<div id="education" class="container_contents container_contents_3e">
-				<h2 class="container_contents_header"><img src="https://static.igem.org/mediawiki/2016/5/59/T--Tokyo_Tech--Education_String.png" /></h2>
-				<div id="education_overview" class="3e_contents">
-					<h3 class="3e_contents_header">Overview</h3>
-					<p class="normal_text">After exchanging opinions with the public in school festivals (including May festival, Suzukake festival and Homecoming day), we found that the knowledge about genetic modification, <span style="font-style:italic;">E. coli</span> and synthetic biology is less well known. Therefore, we gave classes and made YouTube videos to have the public, especially the youth, know them. Using the familiar story of Snow White as a base to our project, many people got interested on it because of the story, but thanks to that they also got interested in synthetic biology. As a result, more and more people got interested in synthetic biology and <span style="font-style:italic;">E. coli</span> because of the plot of Snow White we used which is familiar to the most. Moreover, after the classes we gave, students improved their awareness of genetic modification and <span style="font-style: italic;">E. coli</span>.</p>
-					<p class="normal_text">&nbsp;</p>
-					<p class="normal_text">We worked on the development of an educational tool for the high school students in order to let the youth study science independently and delightfully. It is a card game about TA system and QS. It attracted more of their attention than only talking about the knowledge.</p>
-				</div><!-- /education_overview -->
-				<div id="education_school_visit" class="3e_contents">
+				<h3 class="link"><a href="#1.introduction">&nbsp;&nbsp;&nbsp;1-1. Introduction</a></h3>
-					<h3 class="3e_contents_header">School-visits</h3>
+				<h3 class="link"><a href="#1.summary">&nbsp;&nbsp;&nbsp;1-2. Summary of the Experiment</a></h3>
-					<p class="normal_text">During the May Festival, the Suzukake Festival and the Home-Coming Day, when we talked to the general public, we found out that the <span style="font-style: italic;">E. coli</span> is being rather misunderstood. So in order to solve this misunderstanding, and to provide more people with a more accurate information about <span style="font-style: italic">E. coli</span>, gene reconfiguration, and synthetic biology in general we decided to conduct some School-visits. As a result, we managed to give classes in 6 Schools and to 269 middle and high school students in total. Furthermore, this activity turned out to be a good opportunity to learn how our project impacted in the general public. We can also say that the fact that the ideas we obtained here managed to positively influence our project is a result out of itself.</p>
+				<h3 class="link"><a href="#1.results">&nbsp;&nbsp;&nbsp;1-3. Results</a></h3>
-					<p class="normal_text">The details are organized for each school, so if anyone were to do this kind of lectures in the future, fell free to use this as reference.</p>
+				<h3 class="link"><a href="#1.discussion">&nbsp;&nbsp;&nbsp;1-4. Discussion</a></h3>
+				<h3 class="link"><a href="#1.methods">&nbsp;&nbsp;&nbsp;1-5. Materials and Methods</a></h3>
-					<div id="school_visit_images">
+				<h3 class="link"><a href="#1.construction"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1-5-1. Construction</font></a></h3>
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+				<h3 class="link"><a href="#1.protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1-5-2. Assay Protocol</font></a></h3>
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-						<img src="https://static.igem.org/mediawiki/2016/5/5c/T--Tokyo_Tech--Koshigaya_North.jpg" id="school_visit_koshigaya_kita" class="school_visit_image" />
-						<img src="https://static.igem.org/mediawiki/2016/8/8d/T--Tokyo_Tech--Atsugi_Lecture.jpg" id="school_visit_atsugi" class="school_visit_image" />
-					</div><!-- /school_visit_image -->
-					<script>
-						$(function(){
-							$('.school_visit_image').hover({
-								function(){
-									$this = $(this).
-								},
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-							});
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-					</script>
-				</div><!-- /education_school_visit -->
-				<div id="education_video" class="3e_contents">
-					<h3 class="3e_contents_header">Video</h3>
-					<p class="normal_text">"It is difficult." "I don't get it" "What are you doing?"</p>
-					<p class="normal_text">These are words that the public told us about our activities, which means that the public did not understand our activity well.Therefore, we made intelligible videos to improve this situation. Moreover, the third episode is dubbed into Japanese, English, Chinese and Spanish so that more people will be able to understand. </p>
-					<p class="normal_text">The first episode: What is <span style="font-style: italic;">E. coli</span>?<br />
-<a href="https://youtu.be/VJre_GtDRo4" target="_blank">https://youtu.be/VJre_GtDRo4</a></p>
-					<p class="normal_text">The second episode: How to modify genes? What can we do by modifying the genes of <span style="font-style:italic;">E. coli</span>?<br />
-<a href="https://youtu.be/nJtvFeMDbac" target="_blank">https://youtu.be/nJtvFeMDbac</a></p>
-					<p class="normal_text">The third episode: "Snow White"<br />
-<a href="https://youtu.be/BCej9LzkYlc " target="_blank"> https://youtu.be/BCej9LzkYlc</a></p>
-				</div><!-- /education_video -->
-				<div id="education_card_game" class="3e_contents">
-					<h3 class="3e_contents_header">Card Game</h3>
-					<p class="normal_text">The public will not understand our project unless they experience it.</p>
-					<p class="normal_text">So we made a card game containing two functions so that the public can understand the basis of our project, TA system and Quorum Sensing. Click here to download the kit with the rulebook that we developed through trial and error so everyone can play easily. If you get interested in this card game, you can print it out and play it yourself.</p>
-				</div><!-- /education_card_game -->
-				<div id="education_expert" class="3e_contents">
-					<h3 class="3e_contents_header">Expert [Ogawa Tatsuya]</h3>
-					<p class="normal_text">We noticed that the ways of manifesting and expressing our project are important when we explain our project to the public.</p>
-					<p class="normal_text">He gave us the advice that we should insert a punch line in our project to summarize our project to the audience's satisfaction. We should not only talk about the results but also convince the audience through our explanation.Hence, we should construct the whole concept, thinking what listeners will think after explanation about our project. </p>
-				</div><!-- /education_expert -->
-				<div id="education_summary" class="3e_contents">
-					<h3 class="3e_contents_header">Summary</h3>
-					<p class="normal_text">We raised the public's attention of genetic modification, <span style="font-style:italic;">E. coli</span> and synthetic biology through our activities.
-					<p class="normal_text">Since we found our activities are influential to many people, we want to continue these to inform the utility of synthetic biology to the public even after iGEM. </p>
-				</div><!-- /education_summary -->
-			</div><!-- /education -->
-			<div id="ethics" class="container_contents container_contents_3e">
-				<h2 class="container_contents_header"><img src="https://static.igem.org/mediawiki/2016/9/95/T--Tokyo_Tech--Ethics_String.png" /></h2>
-			</div><!-- /ethics -->
-			<div id="economy" class="container_contents container_contents_3e">
-				<h2 class="container_contents_header"><img src="https://static.igem.org/mediawiki/2016/7/71/T--Tokyo_Tech--Economy_String.png" /></h2>
-				<div id="economy_ovevrview" class="3e_contents">
-					<h3 class="3e_contents_header">Overview</h3>
-					<p class="normal_text">In order to use our project in society, we decided to approach it from a big perspective, economy. Our project includes a basic research, so, we thought that we should work on our project in terms of economy, instead of more specific fields such as environment and medicine, to make our project flexible to the upcoming technology. Among many elements of our project, in this page we focus on TA system.</p>
-					<p class="normal_text">We had a dialogue with experts to think our project from an economic perspective. This dialogue was the big turning point of our project.</p>
-				</div><!-- /economy_overview -->
-				<div id="economy_expert" class="3e_contents">
-					<h3 class="3e_contents_header">Expert [Nakasaki Kiyohiko]</h3>
-					<p class="normal_text">
-					Prof. Nakasaki is a professor on the department of international development engineering at Tokyo Tech. He is researching about bio-refinery, prevention of plant disease by functional compost and DNA sensor of compost maturity. One of his research aims to produce chemical materials efficiently such as L-lactone and ethanol that is biodegradable plastic materials.</p>
-					<p class="normal_text">&nbsp;</p>
-					<p class="normal_text">He showed interest in the fact that TA system inhibits the cell growth of <i>E. coli</i> and can even "resuscitate" it. And also in the protein expression that comes with this mechanism. During the dialogue, we were given with the positive option that continuously repeating the control of protein production can lead to a technology which reduces the risk of the appearance of multiple-drug-resistant bacteria. Therefore, from the view point of economy, we set a goal to control protein production.</p>
-					<p class="normal_text"><span style="font-weight: bold;">After this dialogue,</span> we have done two things to implement the protein production control.</p>
-					<ol type="i">
-						<li><p class="normal_text">ACA Rise and Fall Code<br />
-							<img src="https://static.igem.org/mediawiki/2016/8/8b/T--Tokyo_Tech--ACA_Dwarfs.jpg" width="150px" class="align_left" />
-							The development of a software which adjusts the number of ACA (Adenine-Cytosine-Adenine) sequence in the DNA.</p>
-							<p class="tokyo_tech_clear">&nbsp;</p></li>
-						<li><p class="normal_text">
-						<a href="https://2016.igem.org/Team:Tokyo_Tech/The experiments of creating E. coli mutants which secrete protein extracellularly">The experiments of creating <i>E. coli</i> mutants which secrete protein extracellularly</a></p>
-                                                    <p class="normal_text"> "TA system ~Queen's Caprice~"
-</p>
-							<p class="normal_text">These resulted in big success to obtain protein production control.</p></li>
-					</ol>
-					<p class="normal_text">&nbsp;</p>
-					<p class="normal_text">Moreover, we had a dialogue with Prof. Nakasaki about the application of the technology of TA system. The subjects for the dialogue were mainly DDS (Drug Delivery System) and risk reduction of the multi-drug-resistant bacteria emergence. These lead to technological development in the future, so the details on them are shown on <a>Future work</a>.</p>
-				</div><!-- /economy_expert -->
-			</div><!-- /economy -->
-		</div><!-- /3E -->
-		<!-- 3E up to here -->
+				</div><!-- /contents_menu -->
+			</div><!-- /contents_contents -->
+		</div><!-- /contents -->
+		<div id="1.introduction" class="container">
+			<div id="1.introduction_header" class="container_header">
+				<h2><span>1-1. Introduction</span></h2>
+			</div><!-- /_header -->
+			<div id="introduction_contents" class="container_contents">
+				<p class="normal_text">The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, <span style="font-style: italic;">mazE</span> expression was induced by the addition of IPTG(2 mM) after <span style="font-style: italic;">mazF</span> expression was induced by the addition of arabinose(0.02%). As a result, it was able to resuscitate from a state of being inhibited cell growth. We named this experiment as "Stop & Go" because it was to resuscitate growth from inhibiting cell growth.
+				</p>
+			</div><!-- /introduction_contents -->
+		</div><!-- /introdution -->
+		<div id="1.summary" class="container">
+			<div id="1.summary_header" class="container_header">
+				<h2><span>1-2. Summary of the Experiment</span></h2>
+			</div><!-- /_header -->
+			<div id="summary_contents" class="container_contents">
+				<p class="normal_text">Transformants as shown below was prepared.<br><br>
+                                <h3 id="fig.3-1-2-1-2-1"> </h3>
+                                <p class="normal_text"><span style="font-style: italic;">E. coli</span> A : carrying the Pcon-<span style="font-style: italic;">rbs-gfp</span> (pSB6A1) , Plac-<span style="font-style: italic;">rbs</span> (pSB3K3)<br><br>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f2/T--Tokyo_Tech--3-1-2-2-5-2.png" height="150"><br></div>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style="font-style: italic;">E. coli</span> A</span>
+				</p></div><br><br><br>
+                                <h3 id="fig.3-1-2-1-2-2"> </h3>
+                                <p class="normal_text"><span style="font-style: italic;">E. coli</span> C : carrying the PBAD-<span style="font-style: italic;">rbs</span> (pSB6A1) , Plac-<span style="font-style: italic;">rbs</span> (pSB3K3)<br><br>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/9a/T--Tokyo_Tech--3-1-2-2-5-1.png" height="150"><br></div>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style="font-style: italic;">E. coli</span> C</span>
+				</p></div><br><br><br>
+                                <h3 id="fig.3-1-2-1-2-3"> </h3>
+                                <p class="normal_text"><span style="font-style: italic;">E. coli</span> B : carrying the PBAD-<span style="font-style: italic;">rbs-mazF-tt</span>-Pcon-<span style="font-style: italic;">rbs-gfp</span> (pSB6A1) , Plac-<span style="font-style: italic;">rbs</span> (pSB3K3)<br><br>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/c/ca/T--Tokyo_Tech--3-1-2-2-5-5.png" height="150"><br></div>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style="font-style: italic;">E. coli</span> B</span>
+                                </p></div><br><br><br>
+                                <h3 id="fig.3-1-2-1-2-4"> </h3>
+                                <p class="normal_text"><span style="font-style: italic;">E. coli</span> D : carrying the PBAD-<span style="font-style: italic;">rbs -mazF-tt-</span>Pcon-<span style="font-style: italic;">rbs-gfp</span> (pSB6A1) , Plac-<span style="font-style: italic;">rbs-mazE</span> (pSB3K3)<br><br>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/3/31/T--Tokyo_Tech--3-1-2-2-5-3.png" height="150"><br></div>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style="font-style: italic;">E. coli</span> D</span>
+                                </p></div><br><br>
+                                <p class="normal_text">It has been found that the expression of Plac is larger than that of PBAD. Therefore, we expected that the expression of <span style="font-style: italic;">mazE</span> would be finally larger than the that of <span style="font-style: italic;">mazF</span>.
+Samples were incubated with vigorous shaking at 37℃. When turbidity reached 0.03, 0.02% arabinose was added to induce <span style="font-style: italic;">mazF</span> expression. Two hours after the addition of arabinose, 2 mM IPTG was added to induce <span style="font-style: italic;">mazE</span> expression. The turbidity and Relative Fluorescece Units (RFU) of GFP were measured at several time points.
+					</p>
+			</div><!-- /summary_contents -->
+		</div><!-- /summary -->
+		<div id="1.results" class="container">
+			<div id="1.results_header" class="container_header">
+				<h2><span>1-3. Results</span></h2>
+			</div><!-- /_header -->
+			<div id="1.results_contents" class="container_contents">
+				<p class="normal_text">From the result in <a href="#fig.3-1-2-1-3-1">Fig. 3-1-2-1-3-1</a>, it was found that MazF inhibited cell growth. The <span style="font-style: italic;">mazE</span> expression was induced 2 h after the <span style="font-style: italic;">mazF</span> expression, and about 8 h later, cell growth resumed. Similarly, the RFU of GFP was also resumed(<a href="#fig.3-1-2-1-3-2">Fig. 3-1-2-1-3-2</a>).
+				</p><br>
+                                <h3 id="fig.3-1-2-1-3-1"> </h3>
+				<div align="center"><img src="https://static.igem.org/mediawiki/2016/d/d2/T--Tokyo_Tech--3-1-2-1-3-1.png"><br></div>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-1-3-1 </span> time vs Turbidity (Stop & Go)
+				</p></div><br>
+                                <h3 id="fig.3-1-2-1-3-2"> </h3>
+				<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/0d/T--Tokyo_Tech--3-1-2-1-3-2.png"><br></div>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-1-3-2 </span> time vs RFU of GFP (Stop & Go)
+				</p></div><br>
+			</div><!-- /results_contents -->
+		</div><!-- /results -->
+		<div id="1.discussion" class="container">
+			<div id="1.discussion_header" class="container_header">
+				<h2><span>4. Discussion</span></h2>
+			</div><!-- /_header -->
+			<div id="1.discussion_contents" class="container_contents">
+				<p class="normal_text">　Before starting the experiments, we anticipated that recovery from the growth inhibition by MazF would be observed immediately after the induction of <span style="font-style: italic;">mazE</span> expression. However, it took 8 h for us to observe the recovery.
+				</p>
+			</div><!-- /discussion_contents -->
+		</div><!-- /discussion -->
+		<div id="1.methods" class="container">
-		<div id="future_work" class="container">
+			<div id="1.methods_header" class="container_header">
-			<div id="future_work_header" class="container_header">
+				<h2><span>5. Materials and Methods</span></h2>
-				<h1><span>Future Work</span></h1>
+			</div><!-- /_header -->
-			</div><!-- /future_work_header -->
+			<div id="1.methods_contents" class="container_contents">
-			<div id="future_work_contents" class="container_contents">
+			<div id="1.construction">
-				<h2 align="header">Overview</h2>
+				<div id="1.construction_header">
-				<p class="normal_text">We consider the possibility of the protein production system is controlled by Maz system and signal transfer mechanism. To improve the performance of controlling the production by Maz system, we developed a tool for increasing or decreasing the number of ACA sequence, which is the specific site cleaved by MazF homo dimmer. Maz system.</p>
+					<h3><span>5-1. Construction</span></h3>
-				<ol type="i">
+				</div><!-- /_header -->
-					<li><p class="normal_text">controlling the protein production </p></li>
+				<div id="1.construction_contents">
-					<li><p class="normal_text">selectivity of the target mRNA</p></li>
+					<p class="normal_text">-Strain
-					<li><p class="normal_text">system of communicating with outside </p></li>
+   					All the samples were XL1-Blue strain.<br>
-				</ol>
+                    -Plasmids<br>
-				<p class="normal_text">Combining i. and ii., it is expected that the target protein is obtained effectively and with favorite rate. Furthermore, adding iii., we could design an automatic and advanced information-processing system. In other words, we could design an oscillatory cell-cell communication in which one cell containing man system, receives an extrinsic signal (Maz system is controlled by the type of the signal), then the cell produces the substance which affects other cells.</p>
+				        &nbsp;&nbsp;&nbsp;<a href="#fig.3-1-2-1-2-1"><span style ="font-style : italic">E. coli</span> A</a> : Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br>
-				<p class="normal_text">To increase the selectivity of repressing the protein production by Maz system, we develop a tool of increasing or decreasing the number of ACA sequence named "ACA Dwarfs." This application makes our future work much more realistic than other iGEM teams (see <a href="https://2016.igem.org/Team:Tokyo_Tech/Model">Model page</a>).</p>
+					</p>
-				<p class="normal_text">Furthermore, a lot of vital phenomena are oscillatory. So we could apply this oscillatory system to DDS (Drug Delivery System) or to decrease the risk of the appearance of the multi-drug-resistant strains. </p>
-				<ul type="disc" style="margin-top: 10px;">
-					<li><p class="normal_text">Decrease the risk of the appearance the multi-drug-resistant strains</p>
-						<p class="normal_text">These days, the possible appearance of multi-drug-resistant strain due to excessive use of pesticides is alarming. But spreading a pesticide one by one costs money, so we suggest using the system of controlling the protein production a by Maz system: the production of a pesticide is usually repressed by MazF, but only when the substance delivered from the targeted organism (for example: pheromone), MazF is contradicted by MazE and the pesticide is produced. This oscillatory model can decrease the possibility of the appearance of the multidrug-resistant strains because the pesticide is produced at intervals.</p>
-					</li>
-					<li><p class="normal_text">DDS(Drug Delivery System)</p>
-						<p class="normal_text">Introducing maz system to human cells and orchestrating the human original signal transduction system and Maz system, we design the mechanism that the cell secrets drug depended on the internal condition. For example, when a maz system introduced cell receives the blood glucose level increase, the cell synthesizes the insulin.</p>
-					</li>
-                                       <li><p class="normal_text">Our Quorum Sensing and TA systems might be applicable to control the Intestinal Flora</p>
-                                              <h3 align="header">1.Introduction</h3>
-                                               <p class="normal_text">These days, Intestinal Flora draws a lot of attention because it will greatly affect our health. If the balance of intestinal flora is lost, it will lead into an illness or might even change your personality. It is assumed that intestinal flora consists of communication systems among various bacteria. However, no such communication systems have been found. So, it will contribute to the society to clarify the mechanism of intestinal flora and to find a way of controlling it.</p>
- 												<p class="normal_text">Our project focused on the cell-cell communication through Toxin-Antitoxin system (TA system) and Quorum Sensing (QS), and proved the initial step to explain and control the system of the intestinal symbiosis.</p>
-<p class="normal_text" style="text-align:center;"><a href="javascript:void(0);" onClick="show('modeling_detail');" class="showHidden"><img src="https://static.igem.org/mediawiki/2016/5/54/T--Tokyo_Tech--readmore.png"></a></p>
+					<p class="normal_text"><a href="#fig.3-1-2-1-2-2"><span style ="font-style : italic">E. coli</span> C</a> : PBAD-<span style ="font-style : italic">rbs</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br>
+					</p>
-	<div id="modeling_detail" class="off">
+					<p class="normal_text"><a href="#fig.3-1-2-1-2-3"><span style ="font-style : italic">E. coli</span> B</a></a> : PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)
-        <h3 align="header">2. The results of our project</h3>
+					</p>
- <h4 align="header">2-1. TA System Assay</h4>
- <p class="normal_text">We used Maz system, which is a kind of TA system. MazF (Toxin) inhibits translation by cleaving ACA sequence in mRNA and MazE (Antitoxin) releases MazF inhibition. We showed that Maz system could regulate cell growth when <i>mazE</i> and <i>mazF</i> were alternately expressed in <i>E. coli</i> (see TA system assay pages).</p> </br>
-<h4 align="header">2-2. AHL and AmiE Assay</h4>
+					<p class="normal_text"><a href="#fig.3-1-2-1-2-4"><span style ="font-style : italic">E. coli</span> D</a></a> : PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazE</span> (pSB3K3)<br>
- <p class="normal_text">We indicated that QS of <i>Pseudomonas aeruginosa genes</i>and <i>V. fischeri</i>, and <i>amiE</i>, which selectively degrades AHLs, could work in <i> E. coli</i>. Sometimes, these genes are toxic to <i>E. coli</i>, but this was solved by, for example, tagging <i>ssrA</i> or experimenting at low temperatures (see AHL reporter assay page and AmiE assay page). </p></br>
+					</p>
-<h4 align="header">2-3. Temperature-dependent Promoters</h4>
- <p class="normal_text">We indicated that temperature-dependent promoters were activated strongly at a specific temperature: <i>gfp</i> under Pcold, which is activated at 15°C and under, was much more expressed at 18°C than at 37°C; on the other hand, <i>gfp</i> under Pheat, which is activated at 42°C and over, was more expressed at 37°C than at 28°C. We can say that we can control the expression of the gene under these temperature-dependent promoters by changing temperature.</p></br>
-<h4 align="header">2-4. Modeling</h4>
+                                        <p class="normal_text">PBAD (BBa_I0050) , Plac (BBa_R0010) , Pcon (BBa_R0040) , <span style ="font-style : italic">gfp</span> (BBa_E0040) , <span style ="font-style : italic">rbs</span> (BBa_B0034) , <span style ="font-style : italic">mazF</span> (BBa_K1096002) , <span style ="font-style : italic">tt</span> (BBa_B0015)
-<p class="normal_text"> We simulated our final genetic circuits containing the genes of TA system, QS and temperature-dependent promoters. The simulation showed that the circuits could perform favorably by improving some parts (see Model page and Rhl system assay page). </p>
+                                        </p>
-                                                    <h3 align="header">3. Discussion</h3>
- <p class="normal_text">In conclusion, we proved that the genes of other bacterial QS and temperature-dependent promoters worked in <i>E. coli</i> and that <i>E. coli</i> original TA system gene under expression-inducing promoters regulated the growth in WET experiments. From these results, the simulation showed that <i>E. coli</i> containing TA system gene and QS gene under various expression-inducing promoters communicated with each other. </br>
+				</div><!-- /constrution_contents -->
- Besides, by simulating that the genetic circuits worked properly with the three types of expression-inducing promoters, we suggest that intestinal bacterial growth is controlled by nutritional condition, temperature and intestinal bacterial density. The three types of promoters were the sugar-dependent promoters (PBAD, Plac), the temperature-dependent promoters (Pcold, Pheat) and the AHL-dependent promoters (Prhl, Plux and Plas).
+			</div><!-- /construction -->
- Our project therefore helps to understand the symbiosis of intestinal flora. Furthermore, we are quite sure that we could engineer the <i>E. coli</i> or other bacteria which have function of regulating the symbiosis.</p>
+<br><br><br><br>
+			<div id="1.protocol">
+				<div id="1.protocol_header">
+					<h3><span>5-2. Assay Protocol</span></h3>
+				</div><!-- /_header -->
+				<div id="1.methods_contents">
+					<p class="normal_text">Pre-culture<br>
+. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).<br><br>
+. Incubate with vigorous shaking for 12 h.<br><br><br>
+　　 					Incubation and Assay<br>
+. Measure the turbidity of the pre-cultures.<br><br>
+. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.<br><br>
+. Incubate with vigorous shaking so that turbidity becomes 0.03.<br><br>
+. Add arabinose so that the final concentration becomes 0.02%.<br><br>
+. 2 h after the addition of arabinose, we added IPTG so that the final concentration becomes 2 mM.<br><br>
+. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at the proper time.
+					</p>
+				</div><!-- /protocol_contents -->
+			</div><!-- /protocol -->
+			</div><!-- /methods_contents -->
+		</div><!-- /methods -->
- </div>
-                                                           </li>
-				</ul>
+		<div id="GaS" class="container container_top2">
-				<!-- future work overview up to here -->
+			<h1 align="center">3-1-2-1 Go & Stop</h1>
+		</div><!-- /page_header -->
+		<div id="contents" class="container">
+			<div id="contents_header" class="container_header">
+				<h2><span>Contents</span></h2>
+			</div><!-- /contents_header -->
+			<div id="contents_contents" class="container_contents">
+				<div id="contents_menu">
-			</div><!-- /future_work_contents -->
+				<h3 class="link"><a href="#2.introduction">&nbsp;&nbsp;&nbsp;2-1. Introduction</a></h3>
-		</div><!-- /future_work -->
+				<h3 class="link"><a href="#2.summary">&nbsp;&nbsp;&nbsp;2-2. Summary of the Experiment</a></h3>
+				<h3 class="link"><a href="#2.results">&nbsp;&nbsp;&nbsp;2-3. Results</a></h3>
+				<h3 class="link"><a href="#2.discussion">&nbsp;&nbsp;&nbsp;2-4. Discussion</a></h3>
+				<h3 class="link"><a href="#2.methods">&nbsp;&nbsp;&nbsp;2-5. Materials and Methods</a></h3>
+				<h3 class="link"><a href="#2.construction"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2-5-1. Construction</font></a></h3>
+				<h3 class="link"><a href="#2.protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2-5-2. Assay Protocol</font></a></h3>
-		<div id="Policy_and_Practices" class="container">
+				</div><!-- /contents_menu -->
-		    <div id="policy_and_practice_header" class="container_header">
+			</div><!-- /contents_contents -->
-			<h1><span>3. Policy & Practices</span></h1>
+		</div><!-- /contents -->
-		    </div><!-- /policy_and_practice_header -->
-		    <div class="container_contents">
-			<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 the success in creation of a well-rounded project with the connection between the public and experts, keeping from our narrow view.</p>
-			<p class="normal_text">This page explains the changes that some events had made to our project in chronological order.</p>
-			<img src="https://static.igem.org/mediawiki/2016/8/8d/T--Tokyo_Tech--Integrated_Slide_Figure.jpg" alt="Integrated_Silde_Figure" width="800px" usemap="#practice_map" />
-			<map name="practice_map">
-			    <area class="practice_map_link scroll_link" shape="rect" coords="19,146,183,280" href="#general_public" />
-			    <area class="practice_map_link scroll_link" shape="rect" coords="575,152,775,261" href="#general_public" />
-			    <area class="practice_map_link scroll_link" shape="rect" coords="190,240,358,374" href="#school_visits" />
-			    <area class="practice_map_link scroll_link" shape="rect" coords="467,218,569,339" href="#school_visits" />
-			    <area class="practice_map_link scroll_link" shape="rect" coords="41,394,321,591" href="#dialog" />
-			    <area class="practice_map_link scroll_link" shape="rect" coords="478,420,773,582" href="#dialog" />
-			    <!--
-			    <area shape="default" href="#" />
-			    -->
-			</map>
-			<div id="general_public">
-			    <p class="normal_text">In the school festivals, we did poster presentation. Through the diverse dialogues with the general public about our preliminary idea of several projects, we found out that the public preferred the Snow White project.</p>
-			    <p class="normal_text">Even though the contents of Snow White project are difficult to understand for the public, they still showed great interest in it because it brought a sense of familiarity to them. And this obviously lead to the public's deeper understanding of Snow White project than the others. Thus we determined our project as Snow White.</p>
-			</div><!-- /general_public -->
-			<div id="school_visits">
-			    <p class="normal_text">Then in the classes we gave to the high school students, we were asked why there are only three characters in the story. Accordingly, we found suitable characters and environmental factors to add into the story. </p>
-			</div><!-- /school_visits -->
-			<div id="dialog">
-			    <p class="normal_text">Another question from the students was that representing the story was interesting, but how our project can contribute to the society. This actually inspired us a lot for we started to focus on the consideration of the prospect of our project.</p>
-			</div><!-- /dialog -->
-			<p class="normal_text"><span style="font-weight: bold;">After having discussing with an expert,</span> we obtained the comment that TA system has the potential to be linked to the development of effective technology.</p>
-			<p class="normal_text">However, at present there exit problems when using a TA system to control the protein production.
-We cannot selectively produce only desired proteins, because other proteins also would be produced at the same time.</p>
-			<p class="normal_text"><span style="font-weight: bold;">Then</span> our dry lab used Java to reseach into the development of a software that can adjust the number of ACA base sequences called "ACA Dwarfs" as a solution to the problem.</p>
-		    </div>
-		    <div class="container_contents">
-			<p class="normal_text">&nbsp;</p>
-			<p class="normal_text">In this way, we integrated Human Practices to out project. However, we have done much more activities for Human Practices and show them in detail below.</p>
-			<p class="normal_text">&nbsp;</p>
-			<p class="normal_text">&nbsp;</p>
-		    </div>
-		    <div id="practice_list" class="container_contents">
-			<div class="practice_wrapper">
-			    <div class="practice_left integrated_human">
-				<p class="normal_text" style="color: #66ccff; text-align: center; font-size: 32px;">Human Practices</p>
-				<h2 align="center">Integrated Human</h2>
-			    </div>
-			    <div class="practice_center" width="4%"></div>
-			    <div class="practice_right practices">
-				<p class="normal_text" style="color: #66ccff; text-align: center; font-size: 32px;">Our Project</p>
-			    <h2 align="center">Practices</h2>
-			    </div>
-			</div>
-			<div class="practice_wrapper">
-			    <div class="practice_left integrated_human integrated_human_contents">
-				<a class="integrated_human_header" href="https://2016.igem.org/Team:Tokyo_Tech/Collaborations#may_festival">Public Engagement / Dialogue<br />May Festival</a>
-				<p class="normal_text">At the school festival of University of Tokyo, the public (parents and friends of students at University of Tokyo, and people who hung around the festival) advised on our project in a poster session.  </p>
-				<p class="normal_text">Among the projects, the ones with approachable themes were quite popular.</p>
-			    </div>
-			    <div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="54px" /></div>
-			    <div class="practice_right practices practices_contents">
-				<p class="integrated_human_header">Settle on "Snow White" from prospective the projects</p>
-				<p class="normal_text">Snow White is a story almost everyone knows, and people can understand the story pretty well. Moreover, it is familiar to the public and promotes a better understanding of <span style="font-style:italic;">E. coli</span>, synthetic biology, and our project.</p>
-			    </div>
-			</div>
-			<div class="practice_wrapper">
-			    <div class="practice_left integrated_human integrated_human_contents">
-				<a class="integrated_human_header" href="">Public Engagement / Dialogue<br />Suzukake Festival</a>
-				<p class="normal_text">At the school festival of Tokyo Tech, the public (parents and friends of students at Tokyo Tech, and people who hung around the festival) advised on our project in a poster session. We got the advice that we should put on a play to make our story easier to understand.</p>
-			    </div>
-			    <div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="32px" /></div>
-			    <div class="practice_left practices practices_contents">
-				<p class="normal_text">Let's perform a play during the presentation!</p>
-			    </div>
-			</div>
-			<div class="practice_wrapper">
-			    <div class="practice_left integrated_human integrated_human_contents">
-				<a class="integrated_human_header" href="https://2016.igem.org/Team:Tokyo_Tech/HomecomingDay">Public Engagement / Dialogue<br />
-Homecoming Day</a>
-				<p class="normal_text">This is an annual event held at Tokyo Tech which involves everyone related to Tokyo Tech and local people, here we aimed to get feedback from them by introducing our activities with a poster. However, we were not able to tell the public about science and technology easily, and they had trouble understanding the knowledge.</p>
-			    </div>
-			    <div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="32px" /></div>
-			    <div class="practice_right practices  practices_contents">
-				<ul class="normal_text"><li>Make videos about <span style="font-style: italic;">E. coli</span> and synthetic biology for the public.</li>
-<li>Make an interesting card game for  studying.</li></ul>
-			    </div>
-			</div>
-			<div class="practice_wrapper">
-			    <div class="practice_right integrated_human integrated_human_contents">
-				<a class="integrated_human_header" href="">School-visits<br />
-Ochanomizu University Junior High School</a>
-				<p class="normal_text"><span style="font-weight: bold;">The clases' goal was to obtain feedback on our project from junior high school students and to get our project valued with their own evaluation standards.</span> One student offered the following comment.<br />
-"Don't other characters appear in the story?"
-</p>
-			    </div>
-			    <div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="54px" /></div>
-			    <div class="practice_left practices practices_contents">
-				<p class="integrated_human_header">At the beginning, the characters were three: Snow White, the Queen and the Prince. To make our story closer to the original one, we added the Magic Mirror to the story.</p>
-				<p class="normal_text">Snow White story has several versions. So based on our parts, we designed two  gene circuits to represent different story versions.</p>
-			    </div>
-			</div>
-			<div class="practice_wrapper">
-			    <div class="practice_left integrated_human integrated_human_contents">
-				<a class="integrated_human_header" href="">School-visits
-<br>Tokyo Metropolitan Nishi High School</a>
-				    <p class="normal_text"><span style="font-weight: bold;">The classes' goal was to raise student's interest in iGEM through the explanation of the possibility of genetic modification, gene recombination techniques, and synthetic biology. The other goal of this class was to get feedback on our project from high school students. In the class we focused on the explanation of our project at the time.</span> One student gave the following opinion.</p>
-				    <p class="normal_text">"Certainly, representing Snow White Story is interesting, but can you make your project work for society?"</p>
-			    </div>
-			    <div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="32px" /></div>
-				<div class="practice_right practices practices_contents">
-				    <p class="normal_text">Can we make something socially beneficial?</p>
-				<p class="normal_text">We discussed with ourselves and set up an opportunity to have a dialogue with experts.</p>
-				</div>
-			    </div>
-			    <div class="practice_wrapper">
-				<div class="practice_right integrated_human integrated_human_contents">
-				    <p class="integrated_human_header">Expert Prof. Nakasaki</p>
-				    <p class="normal_text">During the dialogue, He said that repetition of protein production control probably leads to a technology which reduces the risk of the multiple-drug-resistant bacteria emergence.</p>
-				</div>
-				<div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="54px" /></div>
-				<div class="practice_left practices practices_contents"  id="modalopen">
-				    <a>Open</a>
-				</div>
-			    </div>
-			    <!-- model　-->
-			    <div id="modal_background">
-			    <div id="modal_contents"><p class="normal_text">We got the positive opinion that TA system probably leads to a valid technology in the future.</p>
-				<p class="normal_text">However, there are two problems with protein production control using TA system.<br />
+		<div id="2.introduction" class="container">
-(i), You cannot produce target protein selectively; you produce other needless protein simultaneously. <br />
+			<div id="2.introduction_header" class="container_header">
-(ii), Repetition of protein production control leads to sustainable production in one system. However, to take protein from <span style="font-style: italic;">E. coli</span>, you have to destroy <span style="font-style: italic;">E. coli</span>, which makes sustainable production impossible at present.</p>
+				<h2><span>2-1. Introduction</span></h2>
+			</div><!-- /_header -->
+			<div id="2.introduction_contents" class="container_contents">
+				<p class="normal_text">In Experiment 1-2-1, we found that a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what will happen when a toxin is expressed after the antitoxin constitutive expression? Therefore, we conducted the experiment. Since cell growth was resuscitated after cells had grown, we named this experiment, "Go & Stop".
+				</p>
+			</div><!-- /introduction_contents -->
+		</div><!-- /introdution -->
-				<p class="normal_text">We continued the activities to find the clue of two critical problems. We worked on them (i) from a view of modeling, (ii) from a view of experiments, and finally found solutions for the problems.</p>
+		<div id="2.summary" class="container">
+			<div id="2.summary_header" class="container_header">
+				<h2><span>2-2. Summary of the Experiment</span></h2>
+			</div><!-- /_header -->
+			<div id="2.summary_contents" class="container_contents">
+				<p class="normal_text">Transformants as shown below was prepared.
+                                <span style ="font-style : italic">E. coli</span> A : carrying the Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br><br>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f2/T--Tokyo_Tech--3-1-2-2-5-2.png" height="150"><br></div>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style ="font-style : italic">E. coli</span> A</span>
+				</p></div><br><br><br>
-			    <ol type="i">
+                                <p class="normal_text"><span style ="font-style : italic">E. coli</span> C : carrying the PBAD-<span style ="font-style : italic">rbs</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br><br>
-				<li> <p class="normal_text">"ACADwarfs"<br />
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/9a/T--Tokyo_Tech--3-1-2-2-5-1.png" height="150"><br></div>
-				    The development of the software which adjusts the number of ACA (Adenine-Cytosine-Adenine) sequence in the DNA.</p>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style ="font-style : italic">E. coli</span> C</span>
-				    <p class="normal_text">This makes it possible to adjust the inhibition of each protein, that is, you can produce a bigger ration of target protein when MazF works. Moreover, we designed the Java code so that many people can use it.</p></li>
+				</p></div><br><br><br>
-				<li><p class="normal_text">The experiments to make <span style="font-style: italic;">E. coli</span> mutants that secrete protein extracellularly and "TA system ~the Queen's Caprice~"</p>
-				    <p class="normal_text">UV irradiation has being performed to make the mutants that secrete protein extracellularly.</p></li>
-			    </ol>
-			    <p class="normal_text">Using "ACADwarfs" gets the higher grade target protein, which realizes high performance material production system. Additionally, preventing mRNA cleavage of target protein and cleaving mRNA of unnecessary protein can establish sensitive protein labeling.</p>
-			    <p class="normal_text">Furthermore, our research leads to DDS (Drug Delivery System) and reduction of the multi-resistant-bacteria emergence. It should be developed in the future, so the details on them are shown on Future work.</p>
+                                <p class="normal_text"><span style ="font-style : italic">E. coli</span> E : carrying the PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , vector (pSB3K3)<br><br>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/c/ca/T--Tokyo_Tech--3-1-2-2-5-5.png" height="150"><br></div>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style ="font-style : italic">E. coli</span> E</span>
+                                </p></div><br><br><br>
-			</div><!-- /modal_contents -->
-			</div><!-- /modal_background -->
+                                <p class="normal_text"><span style ="font-style : italic">E. coli</span> F : carrying the PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazE</span> (pSB3K3)<br><br>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/3/31/T--Tokyo_Tech--3-1-2-2-5-3.png" height="150"><br></div>
-			<div class="practice_wrapper">
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style ="font-style : italic">E. coli</span> F</span>
-			    <div class="practice_right integrated_human integrated_human_contents">
+                                </p></div><br><br>
-				<a class="integrated_human_header" href="">School-visits<br />
-Kanagawa prefectural Atsugi High School</a>
+                                <p class="normal_text"><span style ="font-style : italic">E. coli</span> G : carrying the PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Pcon-<span style ="font-style : italic">rbs</span>(weak)-<span style ="font-style : italic">mazE</span> (pSB3K3)<br><br>
-				<p class="normal_text">The classes' goal was to raise students' interest and awareness of <span style="font-style: italic">E. coli</span> and to increase the knowledge about synthetic biology and gene ligation techniques.</p>
+                                <div align="center"><img src="https://static.igem.org/mediawiki/2016/3/31/T--Tokyo_Tech--3-1-2-2-5-3.png" height="150"><br></div>
-				<p>The class got the result of an advance in knowledge and enhancement of the students' interest toward genetic modification (However, we could have got better results.) </p>
+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-1 Plasmid diagram of <span style ="font-style : italic">E. coli</span> G</span>
-			    </div>
+                                </p></div><br><br>
-			    <div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="54px" /></div>
-			    <div class="practice_left practices practices_contents">
+                                <p class="normal_text">In order to adjust the amount of expression of <span style ="font-style : italic">mazE</span>, two types of RBS (B0034 and J61117: J61117 is weaker than B0034) was used. Using these plasmids, we tried clarifying stoichiometric relation between A and B by changing the expression of MazE. Samples were incubated with vigorous shaking at 37℃. When turbidity reached 0.03, 0.02% arabinose was added to induce mazF expression. The turbidity and Relative Fluorescece Units (RFU) of GFP were measured at several time points.
-				<p class="normal_text">So far, iGEM team Tokyo_Tech has "told the public about synthetic biology" in various ways with a familiar theme. However, we have not been able to influence all of the people. We thought that the reason relied on our way of communication. Therefore, we decided to ask a science communicator, who is an expert  on telling science to the public.</p>
+					</p>
-			    </div>
+			</div><!-- /2.summary_contents -->
+		</div><!-- /2.summary -->
+		<div id="2.results" class="container">
+			<div id="2.results_header" class="container_header">
+				<h2><span>2-3. Results</span></h2>
+			</div><!-- /_header -->
+			<div id="2.results_contents" class="container_contents">
+				<p class="normal_text">Increase in turbidity and RFU of <span style ="font-style : italic">E. coli</span> G stopped earlier than that of <span style ="font-style : italic">E. coli</span> F(<a href="#fig.3-1-2-2-3-1">Fig. 3-1-2-2-3-1</a> and <a href="#fig.3-1-2-2-3-2">Fig. 3-1-2-2-3-2</a>).
+As a result, turbidity and the RFU of <span style ="font-style : italic">E. coli</span> F in the stationary phase was smaller than that of <span style ="font-style : italic">E. coli</span> G.</p>
+                                <h3 id="fig.3-1-2-2-3-1"> </h3>
+				<div align="center"><img src="https://static.igem.org/mediawiki/2016/2/26/T--Tokyo_Tech--Description_Toxin6.png" width="400px"/>
+				<p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-3-1.</span>Time vs Turbidity (Go &Stop)
+				</p><br>
+                                <h3 id="fig.3-1-2-2-3-2"> </h3>
+				<img src="https://static.igem.org/mediawiki/2016/b/b7/T--Tokyo_Tech--Description_Toxin7.png" width="400px"/>
+				<p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-3-2</span>Time vs RFU of GFP (Go &Stop)
+				</p></div><br>
+                <p class="normal_text">Calculation of the change of RFU of GFP / Turbidity per unit time (translation efficiency) indicates that the expression level of MazE correlated with the translation efficiency(<a href="#fig.3-1-2-2-3-3">Fig. 3-1-2-2-3-3</a>).
+                                <h3 id="fig.3-1-2-2-3-3"> </h3>
+				<div align="center"><img src="https://static.igem.org/mediawiki/2016/e/e7/T--Tokyo_Tech--Description_Toxin8.png" width="400px"/>
+				<p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-3-3</span>translation efficiency of each <span style ="font-style : italic">E. coli.</span>
+				</p><br>
+				</div>
 			</div>
+		</div>
-			<div class="practice_wrapper">
-			    <div class="practice_left integrated_human integrated_human_contents">
-				<a class="integrated_human_header" href="https://2016.igem.org/Team:Tokyo_Tech/Human_Practices/National_museum">National museum, nature and science, Tokyo<br>
-				Science Communicator Mr. Ogawa</a>
-				<p class="normal_text">He told us that only representing the research cannot attract the audience's interest, and cannot get shrewd feedback. Also, he suggested that our story should have a clear conclusion that can tell people something.</p>
-			    </div>
-			    <div class="practice_center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--Tokyo_Tech--Apple.png" width="32px" /></div>
-			    <div class="practice_right practices practices_contents">
-				<p class="normal_text">We learned how to tell science to the public from a science communicator. From his talk, we noticed that we should design the whole project considering what the public will think after explaining our project. </p>
-				<p class="normal_text">In addition, we decided to make our project more attractive, through clarifying our project's goal and setting a clear conclusion.</p>
-			    </div>
-			</div>
-		    </div><!-- /practice_list -->
-		    <div id="summary" class="container_contents">
-			<h2 align="center">Summary</h2>
-			<p class="normal_text">We "went beyond the lab" and developed our project through human practice activities.</p>
-			<p class="normal_text">We have not just reflected the opinions from the dialogue with the public and experts about our project. Firstly, we investigated and discussed the issues on our own. Secondly, in the light of the application of our project, we did the experiments and modeling. Finally, we succeeded in the creation of "ACADwarfs" and "TA system～Queen's Caprice～". Furthermore, we are doing the experiments to make <span style="font-style: italic;">E. coli</span> mutants secreting protein extracellularly. The results will probably lead to further developments of our project. </p>
-			<p class="normal_text">Achievements as mentioned above indicate that we have succeeded in the integrated Human Practice.</p>
-			<h3><a href="https://2016.igem.org/Team:Tokyo_Tech/Collaborations#may_festival">Suzukake Festival (May 14 & 15, 2016)</a></h3>
-			<img src="https://static.igem.org/mediawiki/2016/8/81/T--Tokyo_Tech--May_Festival.png" alt="May Festival" width="200px" style="float: left;" />
-			<p class="normal_text" style="float: left; width: 550px;">The Suzukake Festival, the school festival on the Suzukakedai campus, was held on May 14-15, 2016. We introduced iGEM and our projects to the visitors to let them know about synthetic biology and we received feedback.</p>
-			<div class="tokyo_tech_clear"></div>
-			<h3><a href="https://2016.igem.org/Team:Tokyo_Tech/HomecomingDay">Homecoming Day (May 21, 2016)</a></h3>
-			<img src="https://static.igem.org/mediawiki/2016/6/69/T--Tokyo_Tech--Homecoming_Day.jpg" alt="PHOTO" width="200px" style="float:left;" />
-			<p class="normal_text" style="float: left; width: 550px;">Homecoming Day is an annual event held at Tokyo Tech. We introduced iGEM and synthetic biology to the alumni. We realized that it is hard to explain our research themes and goals to the public clearly. Therefore, we decided to make videos about <i>E. coli</i> and genetic modification. </p>
-			<div class="clear"></div>
-		    </div><!-- /summary -->
-		    <div id="reference" class="container_contents">
-			<h2 align="center">Reference</h2>
-			<!--
-http://catalog.takara-bio.co.jp/PDFS/50_24-27.pdf<br />
-file:///C:/Users/Mako/Documents/iGEM/MazF社会貢献性（single%20protein）.pdf
--->
-		    </div><!-- /reference -->
-		</div><!-- /Policy_and_Practices -->
-		<!-- Policy and Practice up to here -->
+		<div id="2.discussion" class="container">
+			<div id="2.discussion_header" class="container_header">
+				<h2><span>2-4. Discussion</span></h2>
+			</div><!-- /_header -->
+			<div id="discussion_contents" class="container_contents">
+				<p class="normal_text">We found that MazF inhibited cell growth and translation even when there was MazE.
+In addition,these results suggested that there was a relation between the resuscitation and <span style ="font-style : italic">mazE</span> expression. <br><br>
+&nbsp;&nbsp;&nbsp;From results of Experiment1.2.1. and Experiment1.2.2., it was expected that <span style ="font-style : italic">mazEF</span> can repeatedly control cell growth.
-		<div id="Snow_White_Versions" class="container">
-			<div id="Snow_White_Vesions_header" class="container_header">
-				<h1><span>4. Snow White Versions</span></h1>
-			</div><!-- /Snow_White_Versions_header -->
-			<div id="Snow_White_Versions_contents" class="container_contents">
-				<p class="normal_text">The remaking of the fairy tale "Snow White" by the Grimm Brothers has enjoyed a lot of versions since it was published in 1812 in the first edition of their collection Grimms" Fairy Tales. Some elements are common in any Snow White stories. However, because the authors add their original elements or features to the story, it becomes unique. We chose other two versions, <a href="https://2016.igem.org/Team:Tokyo_Tech/Snow_White_Versions">Mirror Mirror" and "A Snow White Christmas"</a> and designed their genetic circuits.
-				</p>
-			<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/74/T--Tokyo_Tech--HP_kairo1.png" width="400px"/></div><br>
-			<div align="center"><img src="https://static.igem.org/mediawiki/2016/d/d1/T--Tokyo_Tech--_HP_kairo2.png" width="400px"/>
-			<p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;"></span>
-			</p><br></div>
-			</div><!-- /Snow_White_Versions_contents -->
+				</p>
-		</div><!-- /Snow_White_Versions -->
+			</div><!-- /discussion_contents -->
-	</div><!-- /main_contents -->
+		</div><!-- /discussion -->
-	<script type="text/javascript">
-		$(function(){
-		    $("#modalopen").click(function(){
+		<div id="2.methods" class="container">
-			//$("body").append('<div id="modal_background"></div>');
+			<div id="2.methods_header" class="container_header">
+				<h2><span>2-5. Materials and Methods</span></h2>
-			modalResize();
+			</div><!-- /_header -->
+			<div id="2.methods_contents" class="container_contents">
-			$("#modal_background, #modal_contents").fadeIn("slow");
+			<div id="construction">
+				<div id="construction_header">
-			$("#modal_background, #modal_contents").click(function(){
+					<h3><span>2-5-1. Construction</span></h3>
-			    $("#modal_contents, #modal_background").fadeOut("slow", function(){
+				</div><!-- /_header -->
-				$('#modal_background').remove() ;
+				<div id="2.construction_contents">
-			    });
+					<p class="normal_text">-Strain<br>
+					All the samples were XL1-Blue strain.<br><br>
-			});
-			$(window).resize(modalResize);
-		     function modalResize(){
-			var w = $(window).width();
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-				"left": ((w - cw)/2) + "px",
-				"top": "50px"
-			});
-		    }
-		});
-	    });
+                    -plasmid<br>
+					&nbsp;&nbsp;&nbsp;<span style ="font-style : italic">E. coli</span> C : PBAD-<span style ="font-style : italic">rbs</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br>
+					</p>
-		/**** Rotate Image *****/
+					<p class="normal_text"><span style ="font-style : italic">E. coli</span> A : Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br>
+					</p>
-		var parent = document.getElementById('images_wrapper');
+					<p class="normal_text">E.coli E : PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , vector (pSB3K3)<br>
-		var string3e = document.getElementById('string_3e');
+					</p>
-		var education = document.getElementById('education_string');
-		var ethics = document.getElementById('ethics_string');
+                                        <p class="normal_text"><span style ="font-style : italic">E. coli</span> G : PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Pcon-<span style ="font-style : italic">rbs</span>(BBa_J61117)-<span style ="font-style : italic">mazE</span> (pSB3K3)
-		var economy = document.getElementById('economy_string');
+					</p>
+					<p class="normal_text"><span style ="font-style : italic">E. coli</span> F : PBAD-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">mazF</span>-<span style ="font-style : italic">tt</span>-Pcon-<span style ="font-style : italic">rbs</span>-<span style ="font-style : italic">gfp</span> (pSB6A1) , Pcon-<span style ="font-style : italic">rbs</span>(BBa_B0034)-<span style ="font-style : italic">mazE</span> (pSB3K3)
+					</p>
+                                        <p class="normal_text">PBAD (BBa_I0050) , Plac (BBa_R0010) , Pcon (BBa_R0040) , <span style ="font-style : italic">gfp</span> (BBa_E0040) , <span style ="font-style : italic">rbs</span> (BBa_B0034) , <span style ="font-style : italic">mazF</span> (BBa_K1096002) , <span style ="font-style : italic">mazF</span> (BBa_K1096001),<span style ="font-style : italic">tt</span> (BBa_B0015) , weak <span style ="font-style : italic">rbs</span> (BBa_J61117)
+                                        </p>
+					</p>
+				</div><!-- /2.constrution_contents -->
+			</div><!-- /2.construction -->
+<br><br><br><br>
+			<div id="2.protocol">
+				<div id="2.protocol_header">
+					<h3><span>2-5-2. Assay Protocol</span></h3>
+				</div><!-- /_header -->
+				<div id="2.protocol_contents">
+					<p class="normal_text">Pre-culture<br>
+. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).<br>
+. Incubate with vigorous shaking for 12 h.<br><br>
+					Incubation and Assay<br>
+. Measure the turbidity of the pre-cultures.<br>
+. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin. <br>
+. Incubate with vigorous shaking so that turbidity becomes 0.03<br>
+. Add arabinose so that the final concentration becomes 0.02%.<br>
+. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at proper times.
+					</p>
+				</div><!-- /protocol_contents -->
+			</div><!-- /protocol -->
+			</div><!-- /methods_contents -->
+		</div><!-- /methods -->
-		var hasFinishedOnload = new Array(0,0,0,0);
+<div id="Queen" class="container container_top2">
+			<h1 align="center">3-1-2-3 <span style ="font-style : italic">mazEF</span> System Assay on the LB Agar Plate</h1>
-		var string3eWidth, string3eHeight,
+		</div><!-- /page_header -->
-			educationWidth, educationHeight,
-			ethicsWidth, ethicsHeight,
+		<div id="contents" class="container">
-			economyWidth, economyHeight;
+			<div id="contents_header" class="container_header">
+				<h2><span>Contents</span></h2>
-		var educationRatio;
+			</div><!-- /contents_header -->
-		var ethicsRatio;
+			<div id="contents_contents" class="container_contents">
-		var economyRatio;
+				<div id="contents_menu">
-		var theta;
-		var rotateRadius;
-		var image3e = new Image();
-		image3e.onload = function(){
-			string3eWidth = image3e.width;
-			string3eHeight = image3e.height;
-			hasFinishedOnload[0] = 1;
-		};
-		image3e.src = 'https://static.igem.org/mediawiki/2016/9/9a/T--Tokyo_Tech--3E_String.png';
-		var imageEducation = new Image();
-		imageEducation.onload = function(){
-			educationWidth = imageEducation.width;
-			educationHeight = imageEducation.height;
-			hasFinishedOnload[1] = 1;
-		};
-		imageEducation.src = 'https://static.igem.org/mediawiki/2016/5/59/T--Tokyo_Tech--Education_String.png';
-		var imageEthics = new Image();
-		imageEthics.onload = function(){
-			ethicsWidth = imageEthics.width;
-			ethicsHeight = imageEthics.height;
-			hasFinishedOnload[2] = 1;
-		};
-		imageEthics.src = 'https://static.igem.org/mediawiki/2016/9/95/T--Tokyo_Tech--Ethics_String.png';
-		var imageEconomy = new Image();
-		imageEconomy.onload = function(){
-			economyWidth = imageEconomy.width;
-			economyHeight = imageEconomy.height;
-			hasFinishedOnload[3] = 1;
-		};
-		imageEconomy.src = 'https://static.igem.org/mediawiki/2016/7/71/T--Tokyo_Tech--Economy_String.png';
-		/** Embed Image Function **/
-		function embedImage(place, image, url){
-			place.innerHTML = '<a href="#' + url + '" class="scroll_link"><img src="' + image.src + '" /></a>';
-		}
-		embedImage(string3e, image3e, '');
-		embedImage(education, imageEducation, 'education');
-		embedImage(ethics, imageEthics, 'ethics');
-		embedImage(economy, imageEconomy, 'economy');
-		function rotateImages(){
-			var string3eCenterX = parent.offsetLeft + parent.offsetWidth / 2;
-			var string3eCenterY = parent.offsetTop + string3eHeight / 2 + rotateRadius;
-			parent.style.height=rotateRadius*3 + 'px';
-			string3e.style.left = (string3eCenterX - string3eWidth/2) + 'px';
-			string3e.style.top = (string3eCenterY - string3eHeight/2) +'px';
-			var educationLeft = string3eCenterX - educationRatio * rotateRadius * Math.sin(theta) - educationWidth/2;
-			var educationTop = string3eCenterY - rotateRadius * Math.cos(theta) - educationHeight/2;
-			var ethicsLeft = string3eCenterX - educationRatio * rotateRadius * Math.sin(theta + Math.PI*2/3) - ethicsWidth/2;
-			var ethicsTop = string3eCenterY - rotateRadius * Math.cos(theta + Math.PI*2/3) - ethicsHeight/2;
-			var economyLeft = string3eCenterX - educationRatio * rotateRadius * Math.sin(theta + Math.PI*4/3) - economyWidth/2;
-			var economyTop = string3eCenterY - rotateRadius * Math.cos(theta + Math.PI*4/3) - economyHeight/2;
-			education.style.left = educationLeft + 'px';
-			education.style.top = educationTop + 'px';
-			ethics.style.left = ethicsLeft + 'px';
-			ethics.style.top = ethicsTop + 'px';
-			economy.style.left = economyLeft + 'px';
-			economy.style.top = economyTop + 'px';
-			theta -= 0.002;
-		}
-		function callRotateImagesFunction(){
-			setInterval(rotateImages, 10);
-		}
-		function onloadLoop(){
-			var i, bool=1;
-			for(i=0; i<4; i++){
-				bool *= hasFinishedOnload[i];
-			}
-			if(bool === 1){
+				<h3 class="link"><a href="#3.introduction">&nbsp;&nbsp;&nbsp;3-1. Introduction</a></h3>
-				educationRatio = (3*educationWidth) / (4*educationHeight);
+				<h3 class="link"><a href="#3.summary">&nbsp;&nbsp;&nbsp;3-2. Summary of the Experiment</a></h3>
-				ethicsRatio = (3*ethicsWidth) / (4*ethicsHeight);
+				<h3 class="link"><a href="#3.results">&nbsp;&nbsp;&nbsp;3-3. Results & Discussion</a></h3>
-				economyRatio = (3*economyWidth) / (4*economyHeight);
+				<h3 class="link"><a href="#3.methods">&nbsp;&nbsp;&nbsp;3-4. Materials and Methods</a></h3>
+				<h3 class="link"><a href="#3.construction"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3-4-1. Construction</font></a></h3>
-				theta = 0.0;
+				<h3 class="link"><a href="#3.protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3-4-2. Assay Protocol</font></a></h3>
-				//rotateRadius = Math.sqrt(Math.pow(string3eWidth+educationWidth, 2) + Math.pow(string3eHeight+educationHeight, 2)) + 10;
-				rotateRadius = (string3eHeight + educationHeight) / 2 + 20;
+				</div><!-- /contents_menu -->
+			</div><!-- /contents_contents -->
-				callRotateImagesFunction();
+		</div><!-- /contents -->
-				return;
-			}
+		<div id="3.introduction" class="container">
-			setTimeout("onloadLoop()", 10);
+			<div id="3.introduction_header" class="container_header">
-		}
+				<h2><span>3-1. Introduction</span></h2>
+			</div><!-- /_header -->
-		onloadLoop();
+			<div id="3.introduction_contents" class="container_contents">
+				<p class="normal_text">　The control of cell growth by the <span style ="font-style : italic">mazEF</span> system has been shown until the previous sections. In this section, we analyzed whether the “stop & go” experiment can be repeated many times.
+				</p>
+			</div><!-- /introduction_contents -->
-		/**** Scroll Link ****/
+		</div><!-- /introdution -->
-		$(function(){
+		<div id="2.summary" class="container">
-			$('.scroll_link').on('click', function(event){
+			<div id="2.summary_header" class="container_header">
-				event.preventDefault();
+				<h2><span>2-2. Summary of the Experiment</span></h2>
+			</div><!-- /_header -->
-				var $this = $(this);
+			<div id="2.summary_contents" class="container_contents">
-				var linkTo = $this.attr('href');
+				<p class="normal_text">A pSB6A1-based plasmid containing both the PBAD (BBa_I0050) - rbs (BBa_B0034) - <span style ="font-style : italic">mazF</span> (BBa_K1096002) cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac (BBa_R0010) - rbs (BBa_B0034) - <span style ="font-style : italic">mazE</span> (BBa_K1096001) cassette was constructed. These plasmids were co-introduced into <span style ="font-style : italic">E. coli</span>. We prepared LB media containing arabinose (Ara(+)), IPTG (IPTG (+)), both inducers (Ara(+), IPTG(+)), and no inducers (Ara (-), IPTG (-)). We tested whether the <span style ="font-style : italic">E. coli</span> cells formed colonies on above plates by controlling the <span style ="font-style : italic">mazEF</span> system. The procedures are shown below.
-				var $target = $(linkTo);
+				</p>
-				var pos = $target.offset().top;
+				<p class="normal_text">(i) Day1 : Each transformant was streaked onto the Ara(+)-, IPTG(+)-, and <br>
-				$('html,body').animate({scrollTop: pos}, 400);
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ara(-)_IPTG(-)-plates.</p>
-			});
+				<p class="normal_text">(ii) Day2 : The colonies on the above plates were re-streaked onto the Ara(+)_IPTG(+)-plate.
-		});
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Even when no colonies were found on the plates, the streaked areas on day 1 were <br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;scratched by toothpicks and re-streaked.</p>
+				<p class="normal_text">(iii) Day3 : The same procedure was conducted as day 2 except that Ara(+)-plate was used.</p>
+				<p class="normal_text">(iv) Day4 : The same procedure was conducted as day 2.</p>
+				<div align="center"><img src="https://static.igem.org/mediawiki/2016/5/55/T--Tokyo_Tech--3-1-3-2-1.png"><br></div>
-		/**** Image Slider ****/
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-2-1-3-1. Overview of the experiment</span>
-		var imagesUrl = [
-			"https://static.igem.org/mediawiki/2016/d/d7/T--Tokyo_Tech--Toritsu_West_Lecture.jpg",
+				</p></div>
-			"https://static.igem.org/mediawiki/2016/b/b2/T--Tokyo_Tech--Kichijoji_Lecture.jpg",
-			"https://static.igem.org/mediawiki/2016/5/5c/T--Tokyo_Tech--Koshigaya_North.jpg",
+			</div><!-- /2.summary_contents -->
-			"https://static.igem.org/mediawiki/2016/7/74/T-Tokyo_Tech--Atsugi_Card_Game.jpg",
+		</div><!-- /2.summary -->
-			"https://static.igem.org/mediawiki/2016/8/8d/T--Tokyo_Tech--Atsugi_Lecture.jpg",
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+		<div id="3.results" class="container">
+			<div id="3.results_header" class="container_header">
+				<h2><span>3-3. Results & Discussion</span></h2>
+			</div><!-- /_header -->
+			<div id="3.results_contents" class="container_contents">
+				<p class="normal_text">From the result, it was clarified that growth of <span style ="font-style : italic">E. coli</span> cells was repeatedly controlled by expression of <span style ="font-style : italic">mazE</span>(<a href="#fig.3-1-2-3-3-1">Fig. 3-1-2-3-3-1</a> and <a href="#table 3-1-2-3-3-1">Table 3-1-2-3-3-1</a>). The results of this experiment are very useful in our project, and it is expected to lead to new biotechnological applications.</p>
+                                <h3 id="fig.3-1-2-3-3-1"> </h3>
+				<div align="center"><img src="https://static.igem.org/mediawiki/2016/b/b9/T--Tokyo_Tech--3-1-3-3-1.png" height ="450"><br></div>
+				<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-3-3-1 Results of culture that was controlled TA system</span>
+				</p></div><br>
-		function show(idName){
+                                <h3 id="table 3-1-2-3-3-1"> </h3>
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+                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Table 3-1-2-3-3-1 Results of culture that was controlled TA system</span>
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+			<div id="discussion_contents" class="container_contents">
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+				<p class="normal_text">From the above result, it was clarified that growth of <span style ="font-style : italic">E. coli</span> cells was repeatedly controlled by expression of <span style ="font-style : italic">mazE</span>. The results of this experiment are very useful in our project, and it is expected to lead to new biotechnological applications.
-				elem2[0].textContent = 'Hide';
+				</p>
-			}
+			</div><!-- /discussion_contents -->
-		}
+		</div><!-- /discussion -->
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+		<div id="3.methods" class="container">
+			<div id="3.methods_header" class="container_header">
+				<h2><span>3-5. Materials and Methods</span></h2>
+			</div><!-- /_header -->
+			<div id="3.methods_contents" class="container_contents">
+			<div id="construction">
+				<div id="construction_header">
+					<h3><span>3-5-1. Construction</span></h3>
+				</div><!-- /_header -->
+				<div id="3.construction_contents">
+					<p class="normal_text">-Strain</p>
+					<p class="normal_text">All the samples were XL1-Blue strain.</p>
+                    <p class="normal_text">-Plasmids</p>
+					<p class="normal_text"><span style="font-style : italic"><span style ="font-style : italic">E. coli</span> D: PBAD-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">mazF</span> (pSB6A1)　，　Plac-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">mazE</span> (pSB3K3)
+					</p>
+					<p class="normal_text"><span style="font-style : italic">E. coli</span> H : PBAD-<span style ="font-style : italic">rbs</span>-<span style="font-style : italic">mazF</span>(pSB6A1)　，　Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)
+					</p>
+					<p class="normal_text"><span style ="font-style : italic">E. coli</span> I : PBAD-<span style ="font-style : italic">rbs</span> (pSB6A1)　，Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)
+					</p>
+					<p class="normal_text">PBAD : BBa_I0050 , Plac : BBa_R0010 , <span style ="font-style : italic">rbs</span> : BBa_B0034 , <span style ="font-style : italic">mazF</span> : BBa_K1096002 , <span style ="font-style : italic">mazE</span> : BBa_K1096001</p>
+				</div><!-- /2.constrution_contents -->
+			</div><!-- /2.construction -->
+<br><br><br><br>
+			<div id="3.protocol">
+				<div id="3.protocol_header">
+					<h3><span>3-5-2. Assay Protocol</span></h3>
+				</div><!-- /_header -->
+				<div id="3.protocol_contents">
+					<p class="normal_text">1) Making LB agar medium containing arabinose and IPTG.</p>
+					<p class="normal_text">2) <span style ="font-style : italic">E. coli</span> are applied at 3 agar medium (in arabinose, in IPTG, in arabinose and IPTG)</p>
+					<p class="normal_text">3) Overnight culture at 37°C for 24 h</p>
+					<p class="normal_text">4) To confirm TA system, inoculate colonies of <span style ="font-style : italic">E. coli</span> having plasmidⅠ, Ⅱ, Ⅴ at agar medium<br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; containig arabinose and IPTG</p>
+					<p class="normal_text">5) Overnight culture at 37°C for 24 h</p>
+					<p class="normal_text">6) Inoculate colonies of <span style ="font-style : italic">E. coli</span> into agar medium containing arabinose.</p>
+					<p class="normal_text">7) Overnight culture at 37°C for 24 h</p>
+					<p class="normal_text">8) Inoculate colonies of <span style ="font-style : italic">E. coli</span> into agar medium in arabinose and IPTG</p>
+					<p class="normal_text"></p>
+				</div><!-- /protocol_contents -->
+			</div><!-- /protocol -->
+			</div><!-- /methods_contents -->
+		</div><!-- /methods -->
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Difference between revisions of "Team:Tokyo Tech/Toxin Assay/mazEF System Assay"

Revision as of 05:46, 19 October 2016

3-1-2 mazEF system Assay

Contents

3-1-2-1 Stop & Go

Contents

1-1. Introduction

1-2. Summary of the Experiment

1-3. Results

4. Discussion

5. Materials and Methods

5-1. Construction

5-2. Assay Protocol

3-1-2-1 Go & Stop

Contents

2-1. Introduction

2-2. Summary of the Experiment

2-3. Results

2-4. Discussion

2-5. Materials and Methods

2-5-1. Construction

2-5-2. Assay Protocol

3-1-2-3 mazEF System Assay on the LB Agar Plate

Contents

3-1. Introduction

2-2. Summary of the Experiment

3-3. Results & Discussion

3-5. Materials and Methods

3-5-1. Construction

3-5-2. Assay Protocol