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| | <h1 align="center">3-1-0. Overview</h1> | | <h1 align="center">3-1-0. Overview</h1> |
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| − | <div id="contents" class="container"> | + | |
| − | <div id="contents_header" class="container_header">
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| − | <h2><span>Contents</span></h2>
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| − | </div><!-- /contents_header -->
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| − | <div id="contents_contents" class="container_contents">
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| − | <h3 class="link"><a href="#TA">3-1-0. TA system</a></h3>
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| − | <h3 class="link"><a href="#AoM">3-1-1. Adjustment of MazF expression</a></h3>
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| − | <h3 class="link"><a href="#Sys">3-1-2. <span style="font-style : italic">mazEF</span> System Assay</a></h3>
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| − | <h3 class="link"><a href="#SaG"><font size="2.7"> 3-1-2-1. Stop & Go</font></a></h3>
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| − | <h3 class="link"><a href="#GaS"><font size="2.7"> 3-1-2-2. Go & Stop</font></a></h3>
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| − | <h3 class="link"><a href="#CoC">3-1-3. Control of Cell Growth</a></h3>
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| − | <h3 class="link"><a href="#Ref">3-1-4. Reference</a></h3>
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| − | </div><!-- /contents_menu -->
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| − | </div><!-- /contents -->
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| | <div id="TA" class="container"> | | <div id="TA" class="container"> |
| − | <div id="TA_header" class="container_header"> | + | |
| − | <h2><span>3-1-0. TA system</span></h2>
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| − | </div><!-- /_header -->
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| | <div id="TA_contents" class="container_contents"> | | <div id="TA_contents" class="container_contents"> |
| − | <p class="normal_text">TA systems have function of controlling cell growth and are present on the genomic DNA of many microorganisms as well as on plasmid DNAs. We thought that exploiting the ability of TA systems was the best way to trace the story of Snow White, because the story includes death and recovery from death. <br><br>
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| − | Preceding iGEM teams have studied the following TA systems: ccdB-ccdA (iGEM 2014 team ULB-Brussels and iGEM 2013 team UGent), Kid-Kis, Zeta-Epsilon(iGEM 2014 team Wageningen UR) and Hok-Soc(iGEM 2015 team UMaryland). The iGEM 2016 team Tokyo_Tech studied the <i>mazEF</i> system, which is one of the most vitally studied TA systems.<br><br>
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| − | MazF (Toxin) specifically recognizes and degrades ACA sequences of mRNAs, thereby inhibiting translation and cell growth. MazE (Antitoxin for MazF) forms a MazF-MazE heterohexamer and neutralize the toxicity of MazF. If this system is used appropriately, it is possible to manipulate growth of <span style="font-style : italic">E. coli</span> cells as we expect, and wide range of biotechnological application is possible.<br><br>
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| − | </p> | + | <p class="normal_text">The experiments of iGEM 2016 team Tokyo_Tech are divided into Toxin, AHL, AmiE, and temperature-dependent promoter assays. This section describes the toxin assay. Our team studied the<span style ="font-style : italic">mazEF</span> system, which is one of the most studied TA systems. |
| − | <div align="center"><img src="https://static.igem.org/mediawiki/2016/9/96/T--Tokyo_Tech--TA_matome.png" height ="500"><br></div> | + | <p class="normal_text">TA systems are found on the genomic DNA of many bacteria and they play important roles in controlling the cell growth. Preceding iGEM teams have |
| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;"> Fig. 3-1-0-1 . </span> Table. Achievement of TA System in the preceding iGEM | + | studied the following TA systems; <span style ="font-style : italic">ccdB ‐ ccdA</span> (iGEM 2014 team ULB-Brussels and iGEM 2013 team UGent), <span style ="font-style : italic">kid ‐ kis</span>, <span style ="font-style : italic">zeta ‐ epsilon</span> (iGEM 2014 team |
| − | </p></div><br> | + | Wageningen UR) and <span style ="font-style : italic">hok ‐ soc</span> (iGEM 2015 team UMaryland) (<a href="#Table. 3-1-0-1.">Table. 3-1-0-1.</a>). Almost all the iGEM teams dealing with TA systems aimed to use them as a kill switch or substitution for antibiotics.<br> |
| − | | + | <h3 id="Table. 3-1-0-1."> </h3> |
| | + | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;"> Table 3-1-0-1 </span> TA system that has been studied in the preceding iGEM .</span> |
| | + | <div align="center"><img src="https://static.igem.org/mediawiki/2016/c/c4/T--Tokyo_Tech--3-1-0-1-1.png" height="450"><br></div> |
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| | + | <p class="normal_text"> On the other hand, we aimed to study cooperation of toxin and antitoxin in cells. Although UCSF 2012 showed the communication through TA systems including the <span style ="font-style : italic">mazEF</span> system, they did not introduce toxin and antitoxin genes into a single cell and did not show the data on the whole <span style ="font-style : italic">mazEF</span> system. Besides, some preceding teams failed to show enough data on TA systems. Our results show that cell growth can be controlled flexibly by manipulating expression ratio of <span style ="font-style : italic">mazE</span> and <span style ="font-style : italic">mazF</span>, implying new applicability of TA systems to many biotechnological fields and future iGEM projects.</span> <br><br> |
| | + | </p></div> |
| | + | </p></div> |
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| − | <div id="AoM" class="container">
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| − | <div id="AoM_header" class="container_header">
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| − | <h2><span>3-1-1. Adjustment of MazF expression</span></h2>
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| − | </div><!-- /_header -->
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| − | <div id="AoM_contents" class="container_contents">
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| − | <p class="normal_text">MazF has very strong ability to inhibit cell growth. A previous study have shown that such inhibition can not be restored even when MazE antitoxin is highly expressed simultaniouly2). Therefore, as a first experiment, <i>mazF</i> gene was placed under the downstream of PBAD, and optimal inducer (L-arabinose) concentration was examined.
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| − | </p>
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| − | <div align="center"><img src="https://static.igem.org/mediawiki/2016/5/5b/T--Tokyo_Tech--TApl_1.png" height ="180"><br></div>
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| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-0-2.</span></p></div>
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| − | <p class="normal_text">From the result of the experiment 1.1., the optimal arabinose concentration was found to be 0.02%. However, under arabinose concentration of 0.2%, GFP fluorescence intensity fell markedly.</p>
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| − | <div id="Sys" class="container">
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| − | <div id="Sys_header" class="container_header">
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| − | <h2><span>3-1-2. <span style="font-style : italic">mazEF</span> System Assay</span></h2>
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| − | </div>
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| − | <div id="Sys_contents" class="container_contents">
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| − | <h3><span>3-1-2-1. Stop & Go</span></h3>
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| − | <div id="SaG_contents">
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| − | <p class="normal_text">Concentration of arabinose to express the appropriate amount of MazF was confirmed in the previous experiment. Based on the result, we have conducted an experiment whether it is control of growth by using the <i>mazEF</i> system.<br>
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| − | We named this experiment as "Stop & Go" because it was to resume growth from suppression the cell growth.
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| − | </p>
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| − | <div align="center"><img src="https://static.igem.org/mediawiki/2016/c/ca/T--Tokyo_Tech--TApl_2.png" height ="180"><br></div>
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| − | <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-0-3.</span></p></div>
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| − | <p class="normal_text">From the results of the experiment 1.2.1., MazE was induced 2 h after MazE expression, and about 8 h later, cell growth that had stopped was recovered .<br>
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| − | Although the revival took longer than expected, suggesting the strength of MazF as the toxin from this result.</p>
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| − | <br><br><br><br>
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| − | <div id="GaS">
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| − | <h3><span>3-1-2-2. Go & Stop</span></h3>
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| − | <div id="_contents">
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| − | <p class="normal_text">In Experiment 1.2.1, it has been found that growth is inhibited by toxin and resuscitated by antitoxin.<br>
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| − | However, if to express the toxin after expression of antitoxin, we wonder how to behave is growth?<br>
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| − | So if MazF expressed after MazE constitutively expressed, we studied how to grow.<br><br>
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| − | From the results of the Exp.1.2.2, it was found that the larger expression of MazE is, the larger the number of <span style="font-style : italic">E. coli</span> is in the Stationary Phase.<br>
| + | </p> |
| − | Further, it was found that there is a correlation efficiency of translation and expression of MazE.
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| − | </p>
| + | <div id="next_page" class="container container_bottom"> |
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| + | <div id="next_page_contents" class="container_contents"> |
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| + | <p class="normal_text">Jump to <a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay/Adjustment_of_Expression_of_MazF"> Adjustment of MazF Expression</a> page.</a> |
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| − | <div id="CoC" class="container">
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| − | <div id="CoC_header" class="container_header">
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| − | <h2><span>3-1-3. Control of Cell Growth</span></h2>
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| − | </div><!-- /_header -->
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| − | <div id="CoC_contents" class="container_contents">
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| − | <p class="normal_text">In order to further secure the control of cell growth, we conducted assay of TA system on the LB agar plate containing inducer of toxin or antitoxin.<br>
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| − | This experiment was named “TA system ~Queen’s capricious～”, because it seemed that princess control a sleep for snow white,<br><br>
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| − | From the results of the Experiment 1.3., it has been found that A cultured in a medium containing inducer of toxin initially may be able to control the colonization by the presence or absence of IPTG(inducer of antitoxin). <br>
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| − | But <span style="font-style : italic">E. coli</span> cultured in a medium containing inducer of antitoxin initially were unable to control the colonization.<br>
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| − | This result might indicate that function of toxin is not only growth inhibition<br><br><br><br>
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| − | If all the above mentioned results are mobilized, it may become possible to freely control of the growth, make of the story with <span style="font-style : italic">E. coli</span>, product the material production in any timing.
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| − | <div id="Ref" class="container">
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| − | <div id="Ref_header" class="container_header">
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| − | <h2><span>3-1-4. Reference</span></h2>
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| − | </div><!-- /_header -->
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| − | <div id="Ref_contents" class="container_contents">
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| − | <p class="normal_text"><br>1) Yonglong Zhang, Junjie Zhang, Klaus P. Hoeflich, Mitsuhiko Ikura, Guoliang Qing, and Masayori Inouye, MazF Cleaves Cellular mRNAs Specifically at ACA to Block Protein Synthesis in <span style="font-style : italic">Escherichia coli</span>. Molecular Cell, Vol. 12, 913–923.<br>
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| − | 2) Hazan, R., B. Sat, and H. Engelberg-Kulka. <span style="font-style : italic">Escherichia coli mazEF</span>mediated cell death is triggered by various stressful conditions. J. Bacteriol.186:3663–3669.
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