Difference between revisions of "Team:Tokyo Tech/Toxin Assay/Overview"

 
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<h1 align="center">3-1-0. Overview</h1>
 
<h1 align="center">3-1-0. Overview</h1>
 
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<h2><span>Contents</span></h2>
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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">&nbsp;&nbsp;&nbsp;3-1-2-1. Stop & Go</font></a></h3>
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<h3 class="link"><a href="#GaS"><font size="2.7">&nbsp;&nbsp;&nbsp;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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<h2><span>3-1-0. TA system</span></h2>
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<p class="normal_text">TA system has a function of controlling the cell growth present on the genomic DNA of many microorganisms.<br>
 
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Preceding iGEM teams studied on 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).<br>
 
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In iGEM 2016 teamTokyo_Tech, we studied on mazEF system, which is actively studied as the TA system.<br><br>
 
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MazF (Toxin) specifically recognizes and degrades the ACA sequence of mRNA, inhibiting the translation and cell growth. MazE(Antitoxin for MazF) form a MazF&#8208MazE heterohexamer complex, it has a function to neutralize the toxicity of MazF. If you can use it well, it is possible to manipulate growth of <span style="font-style : italic">E. coli</span> in freely, a wide range of applications is expected.<br>
 
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We conducted the following experiment in order to pave the application of further TA system.
 
  
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<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.
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/96/T--Tokyo_Tech--TA_matome.png" height ="500"><br></div>
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<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 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-0-1. </span>Table. Achievement of TA System in the preceding iGEM
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studied the following TA systems; <span style ="font-style : italic">ccdB &#8208; ccdA</span> (iGEM 2014 team ULB-Brussels and iGEM 2013 team UGent), <span style ="font-style : italic">kid &#8208; kis</span>, <span style ="font-style : italic">zeta &#8208; epsilon</span> (iGEM 2014 team
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Wageningen UR) and <span style ="font-style : italic">hok &#8208; 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>
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<p class="nomal_text">MazF (Toxin) specifically recognizes and degrades the ACA sequence of mRNA, inhibiting the translation and cell growth. MazE(Antitoxin for MazF) forms a MazF-MazE heterohexamer complex, which has a function that neutralizes the toxicity of MazF1). If you can use the system well, it is possible to regulate growth of <span style="font-style : italic">E. coli</span> freely, and wide range of applications are expected.<br><br>
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                                <h3 id="Table. 3-1-0-1."> </h3>
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                                <div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Table 3-1-0-1 </span> TA system that has been studied in the preceding iGEM.</span>
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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>
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We conducted the following experiment to pave the application of further TA system.
 
  
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<h2><span>3-1-1.　Adjustment of MazF expression</span></h2>
 
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<p class="normal_text">MazF has very strong ability to inhibit cell growth, it has been reported not restored even to express too expressed antitoxin2). Therefore, to clarify the expression of MazF capable of suppressing growth of <span style="font-style : italic">E. coli</span>, we examined optimal inducer concentration, downstream from the PBAD.
 
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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., when the assay of TA system using genetic circuit above, the optimal arabinose concentration was found to be 0.02%. However, when Arabinose concentration of 0.2%, GFP fluorescence intensity falls markedly.</p>
 
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<h2><span>3-1-2. <span style="font-style : italic">mazEF</span> System Assay</span></h2>
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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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<h3><span>3-1-2-1. Stop & Go</span></h3>
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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 mazEF 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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<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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<h3><span>3-1-2-2. Go & Stop</span></h3>
 
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<p class="normal_text">In Experiment 1.2.1, it has been found that growth is inhibited by toxin and revived 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 A is, the larger the number of <span style="font-style : italic">E. coli</span> is in the Stationary Phase.<br>
 
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Further, it was found that there is a correlation efficiency of translation and expression of MazE.
 
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<h2><span>3-1-3. Control of Cell Growth</span></h2>
 
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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 Escherichia coli, product the material production in any timing.
 
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<h2><span>3-1-4. Reference</span></h2>
 
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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 Escherichia coli. Molecular Cell, Vol. 12, 913–923.<br>
 
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2) Hazan, R., B. Sat, and H. Engelberg-Kulka. Escherichia coli mazEFmediated cell death is triggered by various stressful conditions. J. Bacteriol.186:3663–3669.
 
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Latest revision as of 11:51, 6 November 2016

3-1-0. Overview

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 themazEF system, which is one of the most studied TA systems.

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 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) (Table. 3-1-0-1.). Almost all the iGEM teams dealing with TA systems aimed to use them as a kill switch or substitution for antibiotics.

Table 3-1-0-1 TA system that has been studied in the preceding iGEM.



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 mazEF system, they did not introduce toxin and antitoxin genes into a single cell and did not show the data on the whole mazEF 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 mazE and mazF, implying new applicability of TA systems to many biotechnological fields and future iGEM projects.

Jump to Adjustment of MazF Expression page.