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

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<h1 align="center">3-1-2 <span style="font-style : italic">mazEF</span> system Assay</h1>
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<h1 align="center">3-1-3 Queen’s Caprice (<span style ="font-style : italic">mazEF</span> System Assay on the LB Agar Plate)</h1>
 
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<h3 class="link"><a href="#SaG"><font size="5">1. Introduction</font></a></h3>
 
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<h3 class="link"><a href="#GaS"><font size="5">2. Summary of the Experiment</font></a></h3>
 
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<h3 class="link"><a href="#2.Summary of the Experiment">&nbsp;&nbsp;&nbsp;2-1. Confirming YafO Function as Toxin on Agar plates</a></h3>
 
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<h3 class="link"><a href="#2.Summary of the Experiment">&nbsp;&nbsp;&nbsp;2-2. Toxin-Antitoxin Assay</a></h3>
 
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<h3 class="link"><a href="#GaS"><font size="5">3. Results</font></a></h3>
 
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<h3 class="link"><a href="#2.Results">&nbsp;&nbsp;&nbsp;3-1. Confirming YafO Function as Toxin on Agar plates</a></h3>
 
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<h3 class="link"><a href="#2.Result">&nbsp;&nbsp;&nbsp;3-2. toxin-antitoxin assay</a></h3>
 
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<h3 class="link"><a href="#GaS"><font size="5">4. Discussion</font></a></h3>
 
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<h3 class="link"><a href="#GaS"><font size="5">5. Materials and Methods</font></a></h3>
 
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<h3 class="link"><a href="#2.Materials and Method">&nbsp;&nbsp;&nbsp;5-1. Strain</a></h3>
 
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<h3 class="link"><a href="#2.Materials and Method">&nbsp;&nbsp;&nbsp;5-2. Plasmid</a></h3>
 
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<h3 class="link"><a href="#2.Plasmid"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5-2-1. Assay to Confirm YafO function as toxin on agar plates</font></a></h3>
 
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<h3 class="link"><a href="#2.Plasmid"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5-2-2. toxin-antitoxin assay</font></a></h3>
 
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<h3 class="link"><a href="#2.Materials and Method"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5-3. Assay Protocol</font></a></h3>
 
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<h3 class="link"><a href="#2.Plasmid"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5-3-1. Confirming YafO function as toxin on agar plate</font></a></h3>
 
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<h3 class="link"><a href="#2.Plasmid"><font size="2.7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5-3-2. toxin-antitoxin assay</font></a></h3>
 
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                                <h3 class="link"><a href="#GaS"><font size="5">6. Reference</a></h3>
 
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<div id="1.introduction" class="container">
 
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<div id="1.introduction_header" class="container_header">
 
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<h2><span>1. Introduction</span></h2>
 
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<div id="introduction_contents" class="container_contents">
 
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<p class="normal_text"> Our project, the story of “Snow White” is constructed based on <span style ="font-style : italic">mazEF</span> system, which is one of toxin-antitoxin (TA) system on E. coli genomic DNA. At the same time, we are interested in other TA systems and we carried out assay using <span style ="font-style : italic">yafNO</span> system.
 
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<div id="2. Summary of the Experiment" class="container">
 
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<div id="2.Summary of the Experiment_header" class="container_header">
 
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<h2><span>2.Summary of the Experiment</span></h2>
 
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                                          <h3><span>2-1. Confirming YafO Function as Toxin on Agar plates </span></h3>
 
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<div id="2.Summary of the Experiment_contents" class="container_contents">
 
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<p class="normal_text">We first confirmed YafO function by observing formation of colonies on agar plates. This experiment was carried out using E. coli which can induce YafO expression by arabinose and E. coli without yafO gene. Construction of plasmids used in this experiment is shown in Fig. 2-1-1. We inoculated four types of E. coli differently on agar plates with or without arabinose.
 
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</p><br>
 
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<br>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-1-2-1. </span>
 
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</p></div>
 
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<div id="1.results" class="container">
 
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<div id="1.results_header" class="container_header">
 
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<h2><span>1-3. Results</span></h2>
 
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</div><!-- /_header -->
 
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<div id="1.results_contents" class="container_contents">
 
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<p class="normal_text">It was found from Fig.3-1-3-2 and Fig.3-1-3-3 that MazF inhibited cell growth. MazE was induced 2 h after MazE expression, and about 8 h later, cell growth was recovered that had stopped. From these results, it was suggested that E. coli whose cell growth was inhibited by MazF was able to resuscitate by expression of MazE.
 
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</p><br>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-1-3-1 </span> time vs Turbidity (Stop & Go)
 
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</p></div><br>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-1-3-2 </span> time vs RFU of GFP (Stop & Go)
 
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<div id="1.discussion" class="container">
 
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<div id="1.discussion_header" class="container_header">
 
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<h2><span>4. Discussion</span></h2>
 
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</div><!-- /_header -->
 
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<div id="1.discussion_contents" class="container_contents">
 
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<p class="normal_text">　From these results, it was found that using TA system can resuscitate the inhibited cell growth. It took much time to resuscitate cell growth. It can be attributed to inhibition of protein synthesis by MazF and the formation of MazE-MazF complex. It is necessary for MazE to be combined with MazF so that MazE acts as an antitoxin of MazF. In addition, it is expected that the production speed of MazE declined because of the translation inhibition caused by MazF.
 
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<div id="1.methods" class="container">
 
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<div id="1.methods_header" class="container_header">
 
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<h2><span>5. Materials and Methods</span></h2>
 
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<div id="1.methods_contents" class="container_contents">
 
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<div id="1.construction">
 
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<div id="1.construction_header">
 
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<h3><span>5-1. Construction</span></h3>
 
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<div id="1.construction_contents">
 
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<p class="normal_text">-Strain
 
−
  All the samples were XL1-Blue strain.<br>
 
−
                    -Plasmids<br>
 
−
1) promoter only : PBAD-<span style ="font-style : italic">rbs</span> (pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br>
 
−
</p>
 
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−
<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. 3-1-2-1-5-1 </span>
 
−
</p></div><br>
 
−
 
−
<p class="normal_text">2) GFP : 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>
 
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</p>
 
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−
<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-1-5-2 </span>
 
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</p></div><br>
 
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−
<p class="normal_text">3) MazF , MazE : 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>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-1-5-3 </span>
 
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</p></div><br>
 
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−
<p class="normal_text">4) MazF : 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)
 
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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--3-1-2-2-5-5.png" height="150"><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. 3-1-2-1-5-4 </span>
 
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</p></div><br>
 
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<br><br><br><br>
 
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<div id="1.protocol">
 
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<div id="1.protocol_header">
 
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<h3><span>5-2. Assay Protocol</span></h3>
 
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</div><!-- /_header -->
 
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<div id="1.methods_contents">
 
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<p class="normal_text">Pre-culture<br>
 
−
1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).<br><br>
 
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2. Incubate with vigorous shaking for 12 h.<br><br><br>
 
−
　　 Incubation and Assay<br>
 
−
1. Measure the turbidity of the pre-cultures.<br><br>
 
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2. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.<br><br>
 
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3. Incubate with vigorous shaking so that turbidity becomes 0.03.<br><br>
 
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4. Add arabinose so that the final concentration becomes 0.02%.<br><br>
 
−
5. 2 h after the addition of arabinose, we added IPTG so that the final concentration becomes 2 mM.<br><br>
 
−
6. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at the proper time.
 
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<div id="GaS" class="container container_top2">
 
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<h1 align="center">3-1-2-1 Go & Stop</h1>
 
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</div><!-- /page_header -->
 
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<div id="2.introduction" class="container">
 
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<div id="2.introduction_header" class="container_header">
 
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<h2><span>2-1. Introduction</span></h2>
 
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</div><!-- /_header -->
 
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<div id="2.introduction_contents" class="container_contents">
 
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<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".
 
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<div id="2.summary" class="container">
 
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<div id="2.summary_header" class="container_header">
 
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<h2><span>2-2. Summary of the Experiment</span></h2>
 
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</div><!-- /_header -->
 
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<div id="2.summary_contents" class="container_contents">
 
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<p class="normal_text">A pSB6A1-based plasmid containing both the PBAD (BBa_I0050)-<span style ="font-style : italic">rbs</span> (BBa_B0034)-<span style ="font-style : italic">mazF</span> (BBa_K1096002) and the Pcon (BBa_R0040)-<span style ="font-style : italic">rbs</span> (BBa_B0034)-<span style ="font-style : italic">gfp</span> (BBa_E0040) cassettes was constructed. Furthermore, a pSB3K3-based plasmid containing the Pcon (BBa_R0010)-<span style ="font-style : italic">rbs</span>(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>. In addition, a pSB3K3-based plasmid containing the Pcon-<span style ="font-style : italic">rbs</span>(BBa_J61117)-<span style ="font-style : italic">mazE</span> cassette was constructed, using RBS (BBa_J61117), which is weaker than RBS (BBa_B0034). Using these plasmids, we tried clarifying stoichiometric proportion by changing the expression of MazE with two types of RBS (B0034 and J61117) downstream of Pcon.
 
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</p><br>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-2-1. </span>
 
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</p></div>
 
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<div id="2.results" class="container">
 
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<div id="2.results_header" class="container_header">
 
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<h2><span>2-3. Results</span></h2>
 
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</div><!-- /_header -->
 
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<div id="2.results_contents" class="container_contents">
 
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<p class="normal_text"><span style ="font-style : italic">E. coli</span> encoded <span style ="font-style : italic">mazE</span> which is on downstream of weak RBS(J61117) has more turbidity than <span style ="font-style : italic">E. coli</span> encoded <span style ="font-style : italic">mazE</span> which is on downstream of normal RBS(B0034). Both of those <span style ="font-style : italic">E. coli</span> would reach stationary phase when there is little RFU of GFP. <span style ="font-style : italic">E. coli</span> encoded <span style ="font-style : italic">mazE</span> which is on downstream of normal RBS (B0034) reached almost the same stationary phase as <span style ="font-style : italic">E. coli</span> without TA system</p>
 
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/2/26/T--Tokyo_Tech--Description_Toxin6.png" width="400px"/>
 
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<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)
 
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</p><br>
 
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<img src="https://static.igem.org/mediawiki/2016/b/b7/T--Tokyo_Tech--Description_Toxin7.png" width="400px"/>
 
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<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)
 
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</p></div><br>
 
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                <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.
 
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−
<div align="center"><img src="https://static.igem.org/mediawiki/2016/e/e7/T--Tokyo_Tech--Description_Toxin8.png" width="400px"/>
 
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<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>
 
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</p><br>
 
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</div>
 
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<div id="2.discussion" class="container">
 
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<div id="2.discussion_header" class="container_header">
 
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<h2><span>2-4. Discussion</span></h2>
 
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</div><!-- /_header -->
 
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<div id="discussion_contents" class="container_contents">
 
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<p class="normal_text">From this experimental result, we found that MazF inhibits cell growth and translation even when there is MazE. Additionally, Fig 3-1-2-2-3-3 showed that the more MazE was expressed, the higher the translation efficiency got. Therefore, the result suggests that MazF inhibits translation, and MazE resuscitates the translation inhibition; the resuscitation correlates MazE expression. This experimental result is associated with anamnestic experimental results which indicated that MazE and MazF form a hexamer (MazF2-MazE2-MazF2).
 
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                                The results of Experiment1.2.1. and Experiment1.2.2. seem that using <span style ="font-style : italic">mazEF</span> System can repeat the inhibition of cell growth and translation by a toxin, and resuscitation of cell growth and translation by an antitoxin.
 
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</p>
 
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</div><!-- /discussion_contents -->
 
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</div><!-- /discussion -->
 
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<div id="2.methods" class="container">
 
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<div id="2.methods_header" class="container_header">
 
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<h2><span>2-5. Materials and Methods</span></h2>
 
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</div><!-- /_header -->
 
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<div id="2.methods_contents" class="container_contents">
 
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<div id="construction">
 
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<div id="construction_header">
 
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<h3><span>2-5-1. Construction</span></h3>
 
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</div><!-- /_header -->
 
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<div id="2.construction_contents">
 
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<p class="normal_text">-Strain<br>
 
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All the samples were XL1-Blue strain.<br><br>
 
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−
                    -plasmid<br>
 
−
1) Vector : Pbad-<span style ="font-style : italic">rbs</span>(pSB6A1) , Plac-<span style ="font-style : italic">rbs</span> (pSB3K3)<br>
 
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</p>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-5-1. </span>
 
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</p></div><br>
 
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<p class="normal_text">2) GFP : 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>
 
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</p>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-5-2. </span>
 
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</p></div><br>
 
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<p class="normal_text">3) MazF , MazE(weak) : 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>
 
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</p>
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-5-3. </span>
 
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</p></div><br>
 
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<p class="normal_text">4) MazF + MazE : 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>
 
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/4d/T--Tokyo_Tech--3-1-2-2-5-4.png" height="150"><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. 3-1-2-2-5-4. </span>
 
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</p></div><br>
 
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<p class="normal_text">5) MazF : 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)
 
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<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>
 
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-1-2-2-5-5. </span>
 
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<h3><span>2-5-2. Assay Protocol</span></h3>
 
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<p class="normal_text">Pre-culture<br>
 
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1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).<br>
 
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2. Incubate with vigorous shaking for 12 h.<br><br>
 
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Incubation and Assay<br>
 
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1. Measure the turbidity of the pre-cultures.<br>
 
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2. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin. <br>
 
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3. Incubate with vigorous shaking so that turbidity becomes 0.03<br>
 
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4. Add arabinose so that the final concentration becomes 0.02%.<br>
 
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5. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at proper times.
 
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Revision as of 17:33, 18 October 2016

3-1-3 Queen’s Caprice (mazEF System Assay on the LB Agar Plate)

Contents