Difference between revisions of "Team:Tokyo Tech/Proof"

 
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<h1 align="center">Proof of concept</h1>
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<h1 align="center">Proof of Concept</h1>
                        <div id="page_header_contents" class="container_contents">
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<h2 align="center">Achievement Gold</h2>
<p class="normal_text">-demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept.</p>
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<div id="page_header_contents" class="container_contents">
                        </div><!-- /page_header_contents -->
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<p class="normal_text">Demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept.</p>
 +
</div><!-- /page_header_contents -->
 
</div><!-- /page_header -->
 
</div><!-- /page_header -->
<div id="contents" class="container">
 
<div id="contents_header" class="container_header">
 
<h2><span>Introduction</span></h2>
 
</div><!-- /contents_header -->
 
<p class="normal_text">The concept of this project concludesare the following 4 points.</p>
 
<div id="contents_contents" class="container_contents">
 
<div id="contents_menu">
 
<h3 class="link"><a href="#Pcold">1. New cold induible promoter (Scene1 : The Magic Mirror’s answer)</a></h3>
 
<h3 class="link"><a href="#Rhl">2. Rhl sysem assay (Scene2 : The Queen’s trap)</a></h3>
 
<h3 class="link"><a href="#TA">3. TA system (Scene3 : Snow White's sleep) </a></h3>
 
<h3 class="link"><a href="#AmiE">4. AmiE degrades C12 (Scene4 : The Prince’s rescue)</a></h3>
 
</div><!-- /contents_menu -->
 
</div><!-- /contents_contents -->
 
<p class="normal_text">We are now going to expound on these points sequentially.</p>
 
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<div id="Pcold" class="container">
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<p class="normal_text">Here we indicate that the cold inducible promoter is a promoter that depends mainly on temperature.<br>
+
<p class="normal_text">Here we indicated that the cold inducible promoter is a promoter that depends mainly on temperature.We performed an experiment using <a href="http://parts.igem.org/Part:BBa_K1949001">BBa_K1949001</a>.
a Pcold-gfp was constracted.<br>
+
<br>
<div align="center"><img src="URL"><br></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/e/ee/T--Tokyo_Tech--6-pr-1-1.png" height ="250"><br></div>
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> title
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 6-3-1-1.  </span> Cold inducible promoter <br>
</p></div>
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the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.
<p class="normal_text">In this experiment, each sample was cultivated at 18°C and 37°C, after that their RFU of GFP was measured.<br>
+
</p></div><br>
From the experiment results it was found that the RFU of GFP of the samples cultivated at 18°C was higher than the ones cultivated at 37°C.<br>
+
<p class="normal_text">In this experiment, each sample was cultivated at 18°C and 37°C, after that their RFU of GFP / Turbidity was measured.</p>
From the above result, we are able to show that this promoter is a promoter induced by low temperatures.
+
<p class="normal_text">From the results, we found that the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.</p>
 +
<p class="normal_text">The above results indicate that this promoter is induced at low temperatures.
  
 
</p>
 
</p>
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</div><!-- /Rhl_header -->
 
</div><!-- /Rhl_header -->
 
<div id="Rhl_contents" class="container_contents">
 
<div id="Rhl_contents" class="container_contents">
<p class="normal_text">Here we indicate how we produced the rhl promoter that suited our project best</p><br>
+
<p class="normal_text">Here shows how we changed the rhl promoter to the one that suited our project best. We conducted an experiment using <a href="http://parts.igem.org/Part:BBa_K1949060">BBa_K1949060</a>.
<div align="center"><img src="URL"><br></div>
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</p><br>
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. </span> title
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/1/17/T--Tokyo_Tech--6-pr-2-1.png" height ="250"><br></div>
</p></div>
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 6-3-2-1. </span> Our original improved Prhl(NM) is much more sensitive to C4HSL than Prhl(WT).
<p class="normal_text">A point mutation was inserted in a wild type rhl promoter.<br>
+
</p></div><br>
In the experiment, an AHL reagent was included in the reporter, and then the fluorescence intensity was measured.<br>
+
<p class="normal_text">A single point mutation was inserted in a wild type rhl promoter(<a href="http://parts.igem.org/Part:BBa_R0071">BBa_R0071</a>).</p>
As a result, the mutant promoters Prhl(M) and Prhl(NM), which is stronger than a wild type promoter, were newly obtained.</p>
+
<p class="normal_text">In the experiment, an AHL reagent was included in the reporter, and then we measured the RFU.</p>
<div align="center"><img src="URL"><br></div>
+
<p class="normal_text">As a result, the mutant promoter Prhl(NM), which is stronger than a wild type promoter, was newly obtained.</p>
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. </span> title
+
<div align="center"><img src="https://static.igem.org/mediawiki/2016/f/f3/T--Tokyo_Tech--6-pr-2-2.png" height ="250"><br></div>
</p></div>
+
<div align="center"><p class="caption" style="font-size: 16px;"><span style="font-weight: bold;">Fig. 6-3-2-2. </span> The Signal-to-Noise (SN) ratio of Prhl(NM) is higher than that of past improved Prhl(LR), and Prhl(NM) does not crosstalk to C12 unlike
<p class="normal_text">iGEM 2014 Tokyo_Tech has already improved the rhl promoter. However, comparing the Prhl(NM) to the Prhl(LR), Prhl(NM) did not show any crosstalk with C12 and the SN ratio of Prhl(NM) was higher than that of Prhl(LR). Also, the experiment showed that Prhl(LR) crosswalks with C12 at a specific proportion. If we use it in the recreation of Snow White, and if the promoter crosstalks with C12, it means that the Queen eats the poisoned apple, she herself produced, and dies. So this is not a promoter we can use in our project.<br>
+
</p></div><br>
The graph also shows that Prhl(NM) almost does not have crosstalk with C12.<br>
+
<p class="normal_text">iGEM 2014 team Tokyo_Tech has improved the rhl promoter. However, comparing the Prhl(NM) with the Prhl(LR), the SN ratio of Prhl(NM) was higher than that of Prhl(LR). Also, the graph shows that Prhl(LR) has crosstalk with C12 at a specific proportion. If we use it in the recreation of Snow White, and the  crosstalk between the promoter and C12 will mean that the Queen eats the Poisoned Apple, which she produced herself, and dies. Therefore, this promoter cannot be used in our project.</p>
Base on the above results, we can say that we found a promoter with strong activity, and it does not have crosstalk with C12,  which is the most suitable for our story.
+
<p class="normal_text">The graph also shows that Prhl(NM) almost have no crosstalk with C12.</p>
 +
<p class="normal_text">Based on the results above, we can say that we found a promoter with strong activity, and it almost have no crosstalk with C12,  which is the most suitable one for our story.</p>
  
 
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<div id="TA_contents" class="container_contents">
 
<div id="TA_contents" class="container_contents">
<p class="normal_text">Here we indicate the inhibition of the <span style="font-style : italic">E. Coli</span>´s multiplication and inhibition of translation by MazF and the restarting of them by MazE.<br>
+
<p class="normal_text">Here shows the inhibition of the <span style="font-style : italic">E. coli´</span>s growth and translation by MazF and the resuscitation of them by MazE. We performed an experiment using <a href="http://parts.igem.org/Part:BBa_K1949100">BBa_K1949100</a> and <a href="http://parts.igem.org/Part:BBa_K1949102">BBa_K1949102</a>.
We constructed a circuit that includes mazEF .
+
 
</p><br>
+
<div align="center"><img src="URL"><br></div>
+
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> title
+
</p></div>
+
<p class="normal_text">In this experiment, MazF was expressed by arabinose , and after 2 hours MazE was expressed by IPTG. <br>
+
From the experiment results, it was found that the turbility of the sample without MazE almost didn’t rise at all. However, it was also found that if IPTG is added, and the n MazE is expressed, the <span style="font-style : italic">E. Coli</span> will restart its multiplication.<br>
+
Also, if only the MazF is working, the RFU of GFP barely rise But if MazE is induced, the RFU of GFP will rise. <a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay">(Link: Toxin assay)</a><br>
+
As the result above, it was concluded that MazF interrupts the multiplication of <span style="font-style : italic">E. Coli</span> and translation. However, MazE restarted the multiplication and translation stopped by MazF.<br>
+
Through this experiment, we showed that the TA system functions properly.</p>
+
<div align="center"><img src="URL"><br></div>
+
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> title
+
</p></div>
+
 
 
 +
</p><br>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2016/e/eb/T--Tokyo_Tech--6-pr-3-1.png" height ="250"><br></div>
 +
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 6-3-3-1. </span> After inhibiting cell growth by MazF, it was resuscitated by expression of MazE.
 +
</p></div><br>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2016/2/24/T--Tokyo_Tech--6-pr-3-2.png" height ="250"><br></div>
 +
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 6-3-3-2. </span> After inhibiting expression of GFP by MazF, it was resuscitated by expression of MazE.
 +
</p></div><br>
 +
<p class="normal_text">In this experiment, MazF was expressed by arabinose, and after 2 h MazE was expressed by IPTG. </p>
 +
<p class="normal_text">From the results, it was found that the turbidity of the sample without MazE almost did not rise at all. However, it was also found that if IPTG is added, and the MazE will be expressed, the <span style="font-style : italic">E. coli</span> will recover its growth.</p>
 +
<p class="normal_text">Also, if only the MazF is working, the RFU of GFP barely rise. If MazE is induced, the RFU of GFP will rise. <a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay/Overview"><a href="https://2016.igem.org/Team:Tokyo_Tech/Toxin_Assay/mazEF_System_Assay">Read mazEF system Assay page</a>.
 +
</a></p>
 +
<p class="normal_text">As the result above, we concluded that MazF inhibits the <span style="font-style : italic">E. coli'</span>s growth and translation. However, MazE resuscitates the growth and translation stopped by MazF.</p>
 +
<p class="normal_text">Althrough this experiment, we indicated that the TA system functions properly.</p>
 +
 
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<div id="AmiE" class="container">
 
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<div id="AmiE_header" class="container_header">
 
<div id="AmiE_header" class="container_header">
<h2><span>4. AmiE degrades C12 </span></h2>
+
<h2><span>4. Degredation of C12 by AmiE </span></h2>
 
</div><!-- /AmiE_header -->
 
</div><!-- /AmiE_header -->
 
<div id="AmiE_contents" class="container_contents">
 
<div id="AmiE_contents" class="container_contents">
<p class="normal_text">Here we indicate that the AmiE decomposes AHL selectively.
+
<p class="normal_text">Here it is indicated that AmiE degrades AHL selectively. We performed an experiment using <a href="http://parts.igem.org/Part:BBa_K1949052">BBa_K1949052</a>.
 +
 
 +
</p><br>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2016/7/75/T--Tokyo_Tech--6-pr-4-1.png" height ="250"><img src="https://static.igem.org/mediawiki/2016/9/94/T--Tokyo_Tech--6-pr-4-2.png" height ="250"></div><br>
 +
<p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 6-3-4-1. </span> AmiE degrades C12  &nbsp;
 +
<span style="font-weight: bold;">Fig. 6-3-4-2. </span> AmiE barely degrades C4 <br>
 +
C12 is greatly degraded. On the other hand, the C4 is barely degraded by AmiE.
 
</p><br>
 
</p><br>
<div align="center"><img src="URL"><br></div>
+
<p class="normal_text">In this experiment, it was examined whether AHLs would be degraded after the addition of 3OC12HSL(C12) and C4HSL(C4) molecules into a culture of <span style="font-style : italic">E. coli</span> that expresses AmiE.<br>
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig.  </span> title
+
From the results, it was found that the C12 added into a culture of AmiE expressing <span style="font-style : italic">E. coli</span> is greatly degraded. On the other hand, the C4 added into this culture, is barely degraded.</p>
</p></div>
+
<p class="normal_text">From the above result, we showed that AmiE selectively degraded AHL molecules.
<p class="normal_text">In this experiment, it was examined whether AHL would be degraded after the addition of 3OC12HSL and C4HSL molecules into a culture of <span style="font-style : italic">E. Coli</span> that express AmiE.<br>
+
From the experiment results, it was found that the C12 added into a culture of AmiE expressing <span style="font-style : italic">E. Coli</span> is greatly degraded. On the other hand, the C4 added in this culture, is barely degraded.<br>
+
From the above result, we were able to show that AmiE selectively degrades AHL molecules.
+
 
</p>
 
</p>
  
 
</div><!-- /AmiE_contents -->
 
</div><!-- /AmiE_contents -->
 
</div><!-- /AmiE -->
 
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Latest revision as of 02:58, 20 October 2016

1. New cold induible promoter

Here we indicated that the cold inducible promoter is a promoter that depends mainly on temperature.We performed an experiment using BBa_K1949001.


Fig. 6-3-1-1. Cold inducible promoter
the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.


In this experiment, each sample was cultivated at 18°C and 37°C, after that their RFU of GFP / Turbidity was measured.

From the results, we found that the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.

The above results indicate that this promoter is induced at low temperatures.

2. Rhl system assay

Here shows how we changed the rhl promoter to the one that suited our project best. We conducted an experiment using BBa_K1949060.



Fig. 6-3-2-1. Our original improved Prhl(NM) is much more sensitive to C4HSL than Prhl(WT).


A single point mutation was inserted in a wild type rhl promoter(BBa_R0071).

In the experiment, an AHL reagent was included in the reporter, and then we measured the RFU.

As a result, the mutant promoter Prhl(NM), which is stronger than a wild type promoter, was newly obtained.


Fig. 6-3-2-2. The Signal-to-Noise (SN) ratio of Prhl(NM) is higher than that of past improved Prhl(LR), and Prhl(NM) does not crosstalk to C12 unlike


iGEM 2014 team Tokyo_Tech has improved the rhl promoter. However, comparing the Prhl(NM) with the Prhl(LR), the SN ratio of Prhl(NM) was higher than that of Prhl(LR). Also, the graph shows that Prhl(LR) has crosstalk with C12 at a specific proportion. If we use it in the recreation of Snow White, and the crosstalk between the promoter and C12 will mean that the Queen eats the Poisoned Apple, which she produced herself, and dies. Therefore, this promoter cannot be used in our project.

The graph also shows that Prhl(NM) almost have no crosstalk with C12.

Based on the results above, we can say that we found a promoter with strong activity, and it almost have no crosstalk with C12, which is the most suitable one for our story.

3. TA system

Here shows the inhibition of the E. coli´s growth and translation by MazF and the resuscitation of them by MazE. We performed an experiment using BBa_K1949100 and BBa_K1949102.



Fig. 6-3-3-1. After inhibiting cell growth by MazF, it was resuscitated by expression of MazE.



Fig. 6-3-3-2. After inhibiting expression of GFP by MazF, it was resuscitated by expression of MazE.


In this experiment, MazF was expressed by arabinose, and after 2 h MazE was expressed by IPTG.

From the results, it was found that the turbidity of the sample without MazE almost did not rise at all. However, it was also found that if IPTG is added, and the MazE will be expressed, the E. coli will recover its growth.

Also, if only the MazF is working, the RFU of GFP barely rise. If MazE is induced, the RFU of GFP will rise. Read mazEF system Assay page.

As the result above, we concluded that MazF inhibits the E. coli's growth and translation. However, MazE resuscitates the growth and translation stopped by MazF.

Althrough this experiment, we indicated that the TA system functions properly.

4. Degredation of C12 by AmiE

Here it is indicated that AmiE degrades AHL selectively. We performed an experiment using BBa_K1949052.



Fig. 6-3-4-1. AmiE degrades C12   Fig. 6-3-4-2. AmiE barely degrades C4
C12 is greatly degraded. On the other hand, the C4 is barely degraded by AmiE.


In this experiment, it was examined whether AHLs would be degraded after the addition of 3OC12HSL(C12) and C4HSL(C4) molecules into a culture of E. coli that expresses AmiE.
From the results, it was found that the C12 added into a culture of AmiE expressing E. coli is greatly degraded. On the other hand, the C4 added into this culture, is barely degraded.

From the above result, we showed that AmiE selectively degraded AHL molecules.