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http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T04:00:04Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li>RNA oscillator</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li>RNAScaffold</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li>Light sensor</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li>Modeling</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin"><li>Improving a BioBrick part - Rhodopsin</li></a><br />
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<p align=center><font size=7>Improving a BioBrick part-Rhodopsin</font></p></font><br />
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Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) </font><br />
<p align=center><font size=5>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg width=500></p><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=600></p><br />
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Fig.5 Comparison of normalized GFPuv fluorescence under white light condition and dark condition.<br />
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Sensory rhodopsin : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1C3-<i>Pconst.-SRII-HtrII-EnvZ-PompC-GFP</i><br />
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Negative control : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1A3-<i>PompC-GFP</i><br />
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Positive control : <i>E.coli</i> BL21(DE3)/pSB1A3-<i>PompC-GFP</i> </p><br />
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<BR>As you can see from the result, we obtained a sensory rhodopsin fused by EnvZ that enhanced the down stream expression in the dark and repressed expression under light. For this year's project, we wanted to combine this light sensor with our Hammerhead ribozyme (HHR), so we decided to construct our genetic device. But however, during the process we realized that the downstream element in EnvZ, about 30 bp before the stop codon of EnvZ, was missing in BBa_K769000. Member from last year did not realize this because they were using the sequence of cph8 for sequence analysis, and did not search against the whole EnvZ sequence. We realized this mid-September, so we quickly re-constructed our sensory-rhodopin fused with EnvZ by designing the following primers:<br />
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<p align=center><imsg src=https://static.igem.org/mediawiki/2013/c/cf/Primers2.jpg></p><br />
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The obtained PCR product was ligated into pSB1A3. Pconst(J23100) and RBS(B0034) were added infront of our new sensory rhodopin fused with EnvZ, and double terminator was added (B0015). This plasmid, together with pSB1C3-PompC-RBS-GFP-doubleTerm. was used to transform <i>E. coli</i> (<i>ΔEnvZ</I>). The colony was confirmed by colony PCR, and was pre-cultured for 12 hours. They were then inoculated into 2 mL LB medium. Half of the samples prepared were covered by aluminium foil and the other half were subjected to light for 12 hours.<br />
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<BR>After 12 hours of culturing under light or wrapped in aluminium foil, the OD at 595 nm and GFP fluorescence was measured. The result is shown below:<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg width=600><br />
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<p align=center>Fig.4 Graph showing the result of light illuminated or non-illuminated culture. <i>E. coli</i> (<i>ΔEnvZ</I>) harboring two plasmids: pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm.</p><br />
<BR><br />
<BR><br />
<BR>As you can see, under the light sensoryrhodopsin-EnvZ enhanced GFP expression and under the dark, it repressed GFP expression. This is a light sensor that can enhance gene expression under light suppress it under dark. However, in practice we do not want high background, meaning that we do not want gene expression leakage in the dark. <br />
<BR><br />
<BR>The graph below shows GFP expression comparison with <i>E. coli</i> (<i>ΔEnvZ</I>) harboring ONLY pSB1C3-PompC-RBS-GFP-doubleTerm, without sensory rhodopsin.<br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/15/Graph1.jpg width=600></p><br />
<BR><br />
<p align=center>Fig.5 Graph showing the result of <i>E. coli</i> (<i>ΔEnvZ</I>) harboring pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm under light or dark, and <i>E. coli</i> (<i>ΔEnvZ</I>) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm. </p><br />
<BR><br />
<BR><br />
<i>E. coli</i> (<i>ΔEnvZ</I>) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm is showing a very high background, which is an indication that probably EnvZ histidine kinase is activated by OmpR from another factor inside the cell. If we want to try and strictly control the gene expression, we might have to use <i>E. coli</i> (<i>ΔEnvZ</I>) with OmpR delted mutation.<br />
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Reference<br />
<BR><br />
<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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http://2013.igem.org/Team:Tokyo-NoKoGen/Achievement
Team:Tokyo-NoKoGen/Achievement
2013-09-28T03:51:35Z
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<p align=center><font size=7>Achievements</p></font><br />
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<BR>We have achieved the following criteria:<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1c/Achievement1.jpg width=800></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/9/90/Achievement2.jpg width=800></p><br />
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<li class="IK"><a target="_blank" href="http://www.tuat.ac.jp/~kakusan/index.html"><img src="https://static.igem.org/mediawiki/2013/8/87/池袋研究室.png"></a></li><br />
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<br />
</body><br />
<br />
</html></div>
Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Achievement
Team:Tokyo-NoKoGen/Achievement
2013-09-28T03:48:23Z
<p>Miyake: </p>
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<div id="mymenubar"><br />
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<table><br />
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<br />
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<a href="#modeling"><li><strong></strong></li></a><br />
<a href="#future work"><li><strong></strong></li></a><br />
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<br />
<div id="main"><br />
<br />
<br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=7>Achievements</p></font><br />
<BR><br />
<BR>We have achieved the following criteria:<br />
<BR><br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1c/Achievement1.jpg width=800></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/9/90/Achievement2.jpg width=800></p><br />
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<li class="UI"><a target="_blank" href="http://www.ultizyme.jp/"><img class="UI" src="https://static.igem.org/mediawiki/2013/4/47/アルティザイム・インターナショナル.jpg" ></a></li><br />
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<li class="LN"><a target="_blank" href="http://lne.st/"><img class="LN" src="https://static.igem.org/mediawiki/2013/2/2a/リバネス.png"></a></li><br />
<li class="IR"><a target="_blank" href="http://www.ikedarika.co.jp/english/"><img class="IR" src="https://static.igem.org/mediawiki/2013/b/b8/池田理化.gif"></a></li><br />
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<br />
<li class="NK"><a target="_blank" href="http://www.tuat.ac.jp/en/index.html"><img class ="NK"src="https://static.igem.org/mediawiki/2013/7/76/東京農工大学.png"></a></li><br />
<li class="ST"><a target="_blank" href="http://www.tuat.ac.jp/~tanpaku/"><img src="https://static.igem.org/mediawiki/2013/5/5a/早出・津川研究室.png" ></a></li><br />
<li class="IK"><a target="_blank" href="http://www.tuat.ac.jp/~kakusan/index.html"><img src="https://static.igem.org/mediawiki/2013/8/87/池袋研究室.png"></a></li><br />
</div><br />
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</body><br />
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</html></div>
Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Achievement
Team:Tokyo-NoKoGen/Achievement
2013-09-28T03:47:16Z
<p>Miyake: </p>
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<p align=center><font size=6>Achievement</p></font><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Achievement
Team:Tokyo-NoKoGen/Achievement
2013-09-28T03:45:35Z
<p>Miyake: </p>
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Achievement
Team:Tokyo-NoKoGen/Achievement
2013-09-28T03:44:36Z
<p>Miyake: </p>
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<p align=center><font size=6>Achievement</p></font><br />
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Miyake
http://2013.igem.org/File:Achievement3.jpg
File:Achievement3.jpg
2013-09-28T03:42:45Z
<p>Miyake: </p>
<hr />
<div></div>
Miyake
http://2013.igem.org/File:Achievement2.jpg
File:Achievement2.jpg
2013-09-28T03:41:19Z
<p>Miyake: </p>
<hr />
<div></div>
Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Achievement
Team:Tokyo-NoKoGen/Achievement
2013-09-28T03:40:11Z
<p>Miyake: </p>
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<td class="home"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen"><span class="home"></span></a></td><br />
<br />
<td class="project"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Project"><span class="project"></span></td><br />
<br />
<td class="team"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Team"><span class="team"></span></a></td><br />
<br />
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<br />
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<br />
<td class="humanpractice"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice"><span class="humanpractice"></span></a></td> <br />
<br />
<td class="achievement1"><div class="achievement1"></div></td><br />
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<br />
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<br />
<div id="index"><br />
<br />
<br />
<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<a href="#overview"><li><strong></strong></li></a><br />
<a href="#modeling"><li><strong></strong></li></a><br />
<a href="#future work"><li><strong></strong></li></a><br />
<li><strong></strong></li><br />
<li><strong></strong></li><br />
<li><strong></strong></li><br />
<li><strong></strong></li><br />
<li><strong></strong></li><br />
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<br />
</div><br />
<br />
<div id="main"><br />
<br />
<br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=6>Achievement</p></font><br />
<BR><br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1c/Achievement1.jpg></p><br />
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</html></div>
Miyake
http://2013.igem.org/File:Achievement1.jpg
File:Achievement1.jpg
2013-09-28T03:38:28Z
<p>Miyake: </p>
<hr />
<div></div>
Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/scaffold
Team:Tokyo-NoKoGen/scaffold
2013-09-28T03:18:47Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
<br />
<link rel="stylesheet" type="text/css" href="Tokyo-NoKoGen.css"><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="wrapper"><br />
<br />
<div id="title_logo"> <br />
<br />
<br />
<a href="https://2013.igem.org"><img class="iGEM" src="https://static.igem.org/mediawiki/2013/5/58/IGEM.png"></a> <br />
</div><br />
<br />
<div id="mymenubar"><br />
<br />
<table><br />
<tbody><br />
<tr><br />
<td class="home"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen"><span class="home"></span></a></td><br />
<br />
<td class="project1"><div class="project1"></div></td><br />
<br />
<td class="team"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Team"><span class="team"></span></a></td><br />
<br />
<td class="biobrick"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Parts"><span class="biobrick"></span></a></td><br />
<br />
<td class="notebook"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Notebook"><span class="notebook"></span></a></td><br />
<br />
<td class="humanpractice"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice"><span class="humanpractice"></span></a></td> <br />
<br />
<td class="achievement"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Achievement"><span class="achievement"></span></a></td><br />
</tbody><br />
</table> <br />
<br />
</div><br />
<br />
<div id="index"><br />
<br />
<br />
<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<li><a href="#Introduction"><strong>Introduction</strong><a><br />
<ul><br />
<li><a href="#Background">Background</a></li><br />
<li><a href="#Objective">Objective</a></li><br />
</ul><br />
</li> <br />
<li><a href="#Method"><strong>Method</strong><a><br />
<ul><br />
<li><a href="#split GFP">split GFP</a></li><br />
<li><a href="#RNA scaffold">RNA scaffold</a></li><br />
<li><a href="#split GFP + RNA scaffold">split GFP + RNA scaffold</a></li><br />
</ul><br />
</li> <br />
<li><a href="#Evaluation"><strong>Evaluation</strong><a><br />
<ul><br />
<li><a href="#Result">Result</a></li><br />
<li><a href="#Discussion">Discussion</a></li><br />
</ul><br />
</li><br />
<li><a href="#Future work"><strong>Future work</strong></a></li><br />
<br />
</ul><br />
<br />
<ul id="contents2"><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li>RNA oscillator</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li>RNAScaffold</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li>Light sensor</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li>Modeling</li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin"><li>Improving a BioBrick part - Rhodopsin</li></a><br />
</ul><br />
<br />
</div><br />
<br />
<div id="main"><br />
<font size=5><br />
<p style="line-height:110%"> <br />
<BR><br />
<BR><br />
<p align=center><font size=7>RNA Scaffold</font></p><BR><BR><br />
<p align=center><strong><font size=6><ins><h1 id="Introduction">Introduction</h1></ins></font></strong></p><BR><br />
</p><br />
<strong><font size=6 id="Background">Background</font></strong><BR><BR><br />
<br />
<p style="line-height:110%"> <br />
<strong><font size=5>SplitGFP</font></strong><BR><br />
Split GFP is split into two fragments between amino acid residue 158 and 159, FA (N-terminal domain) and FB (C-terminal domain). FA alone can’t emit fluorescence. Equally, FB alone can’t emit fluorescence. When FA and FB co-express and meet, the fragments associate and restore fluorescence. Split GFP is used to research RNA detection and localization <I>in vivo</I> by fusion with RNA binding proteins.<BR><BR><br />
<br />
</p><br />
<br />
<p style="line-height:110%"> <br />
<strong><font size=5>Aptamer</font></strong><BR><br />
Aptamers are nucleic acid or peptide molecules, that bind to a specific target molecule. There are many target molecules, for example enzyme, acceptor, virus protein, organic molecule, and so on. RNA aptamer requires a secondary structure formation (hairpin-loop, G-quartet, pseudo knot and bulge loop) to bind to a specific target molecule. RNA aptamers are useful in bio-engineering, because they are easier than proteins for mutation and prediction of secondary structure. In addition, RNA aptamers don’t need to be translated and they work rapidly.<br />
</p><br />
<BR><br />
<BR><br />
<br />
<strong><font size=5>MS2 and PP7</font></strong><BR><br />
<p style="line-height:110%"> MS2 and PP7 are one of the single strand RNA phage coat proteins. RNA aptamer sequences and structures to recognize MS2 and PP7 coat protein have been determined.<br />
</p><br />
<BR><br />
<BR><br />
<br />
<strong><font size=5>Previous work</font></strong><BR><br />
<p style="line-height:110%"> <br />
Camille J. Delebecque and his colleagues have reported organization of bacterial metabolism <I>in vivo</I> by using an RNA scaffold1. They used PP7 and MS2 fused with split GFP to evaluate the scaffold, consists of PP7 and MS2 aptamer domains, whether PP7 and MS2 proteins assemble. They succeeded in assemble PP7 and MS2 proteins. <BR><br />
2012 ZJU-China team registered parts (BB_K73800, BB_K73804, BB_K73805), PP7 and MS2 fused with split GFP which bind to PP7 and MS2 aptamer domains respectively. FA and FB emit fluorescence when they meet. Therefore, when a scaffold containing PP7 and MS2 aptamer domains are expressed, FA and FB come closer and emit fluorescence due to two fusion protein bind to each aptamers.<br />
</p><br />
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<BR><BR><BR><br />
<br />
<strong><font size=6 id="Objective">Objective</font></strong><br />
<br />
<BR><BR><BR><br />
<br />
<strong><font size=5><ins>Functionalization of protein using RNA without translation</ins></font></strong><br />
<BR><br />
<br />
<p style="line-height:110%"> <br />
Tokyo-NoKoGen decided to use these parts to detect an RNA fragment that is produced when HHR causes self-cleavage. There is a complementary sequence to RNA scaffold in HHR, therefore one aptamer does not form secondary structure alone. Only when HHR causes self-cleavage and release the scaffold, the scaffold can form both aptamer domains and result in emitting fluorescence (Fig. 1). On the other hand, when HHR doesn’t cause self-cleavage and release the scaffold, the scaffold can’t form one aptamer domains and result in no fluorescence. <BR><br />
So we will regulate GFP fluorescence by RNA, and the response is faster than protein regulation because it doesn’t need to translate. In addition, we can regulate automatically to use RNA oscillation based on HHR.<br />
</p><br />
<BR><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/a/a1/SplitGFP_aptamer.jpg", height=360, width=480><BR><br />
Fig. 1 How is fluorescence emitted when HHR causes self-cleavage.<br />
</p><BR><BR><BR><br />
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<p align=center><strong><font size=6 id="Method"><ins>Method</ins></font></strong></p><BR></span><br />
<span style="border-bottom:double 1px #000000";><strong>Construction of Parts</strong></span><BR><br />
<p style="line-height:110%"> <br />
Ⅰ Construction of split GFP fused with MS2 and PP7 proteins<BR><br />
<br />
Ⅱ Construction of RNA scaffold<BR><br />
<br />
Ⅲ Construction of split GFP + RNA scaffold<BR><BR><br />
<br />
</P><br />
<br />
<br />
<strong><font size=5 id="split GFP">Ⅰ Construction of split GFP fused with MS2 and PP7 proteins</font></strong><br />
<BR><BR><br />
<p style="line-height:110%"> <br />
1) We used synthesized sequences of split GFP fused with MS2 (FA-linker-MS2) and split GFP fused with PP7 (FB-linker-PP7) respectively, reference of BioBrick part BBa_K738004 (FA-2X-MS2) and BBa_K738005 (FB-2X-PP7) (ZJU-China 2012). Overlap extension PCR was performed to connect the synthesized fragments.<BR><BR><br />
<br />
2-1) The sequence of FB-linker-PP7 was digested at <I>Eco</I>RI and <I>Spe</I>I sites, and BBa_B0015 (double terminator) was digested at <I>Eco</I>RI and <I>Xba</I>I sites, followed by ligation (Fig.2). The region of FB-linker-PP7 and double terminator was amplified by PCR using primers; forward primer has <I>Xba</I>I site and RBS. pSB1A3 and the assembled sequence was digested with <I>Xba</I>I and <I>Spe</I>I (Fig.3).<BR><br />
</P><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/6/63/Fig2.jpg", height=315, width=420><BR><br />
Fig.2 Assemble of double terminator downstream of FB-linker-PP7</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/c/c1/Fig.3.jpg", height=315, width=420><BR><br />
Fig.3 Assemble of RBS upstream of FB-linker-PP7</p><br />
<br />
<p style="line-height:110%"> <br />
2-2) The region of constitutive promoter and RBS in BBa_K317026 (Tokyo-NoKoGen 2010) was amplified by PCR using two primers. The sequence for FA-linker-MS2 constructed in 1) was amplified by PCR. PCR products were ligated into pSB1C3 vector (Fig.4).<BR><BR><br />
</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/6/68/Fig.4.jpg", height=315, width=420><BR><br />
Fig.4 Assemble of constitutive promoter and RBS upstream of FA-linker-MS2</p><BR><BR><br />
<br />
<p style="line-height:110%"> <br />
3) The parts constructed in 2-1, 2-2) were assembled by using digestion with a restriction enzyme. This fragment was inserted into pSB1A3 vector, followed by transformation in <I>E. coli</I> (DH5α) (Fig.5).<BR><br />
</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/6/63/Fig5.jpg", height=315, width=420><BR><br />
Fig.5 Construction of split GFP fused MS2/PP7 proteins</p><BR><BR><br />
<br />
<strong><font size=5 id="RNA scaffold">Ⅱ Construction of RNA scaffold</font></strong><br />
<BR><BR><br />
<br />
<p style="line-height:110%"> <br />
4) BBa_K1053111 (Tokyo-NoKoGen 2013) for RNA scaffold-DT containing MS2 and PP7 aptamer domains, and we chose BBa_J23100 (Berkeley 2006) for constitutive-high promoter as its promoter.<BR><br />
<br />
5) The former plasmid was digested at <I>Xba</I>I and <I>Pst</I>I site, the later plasmid was digested at <I>Spe</I>I and <I>Pst</I>I sites. The digested parts were ligated together. It is our new BioBrick part, BBa_K1053110 (Fig.6). After this part was inserted in pSB1A3, it was transformed in <I>E. coli</I> (DH5α).<BR><br />
</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/9/9d/Fig6.jpg", height=315, width=420><BR><br />
Fig.6 Construction of RNA scaffold</p><BR><BR><br />
<br />
<strong><font size=5 id="split GFP + RNA scaffold">Ⅲ Construction of split GFP + RNA scaffold</font></strong><br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
Each parts constructed in 3) and 5) was digested and ligated (Fig.7), followed by transformation in <I>E. coli</I> (DH5α).<br />
</p><br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/d/d9/Fig7.jpg", height=315, width=420><BR><br />
Fig.7 Assemble of split GFP and RAN scaffold</p><BR><BR><br />
<br />
<p align=center><strong><font size=6 id="Evaluation"><ins>Evaluation</ins></font></strong></p><BR><BR><br />
<br />
<p style="line-height:110%"> <br />
Three transformants (shown below in Fig.10) were inoculated in 3 mL LB medium with relative resistances, and incubated in 37 °C at 150 rpm. After the pre-cultured for over 12 h, the main cultivation was done in 3 mL LB medium with relative resistances inside a L form tube. The experiments were started at cell density at 600 nm (OD<SUB>600</SUB>) of about 0.1. The GFP fluorescence intensity (FI) and cell density (OD<SUB>600</SUB>) were taken, periodically at 2 h intervals for 12 h by Plate Reader.<BR><br />
</p><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/e/eb/Fig8.jpg", height=315, width=420><BR><BR><br />
Fig.8 Evaluation of the FI/OD<SUB>600</SUB> on split GFP and RNA scaffold in <I>E. coli</I></p><BR><BR><BR><br />
<br />
<p align=center><img src="https://static.igem.org/mediawiki/2013/3/30/E.jpg", height=450, width=600><BR><BR><br />
Fig.10 Three transfomants; (A)only RNA scaffold, (B)only split GFP fused MS2/PP7 proteins<BR>(C)RNA scaffold + split GFP.</p><BR><BR><BR><br />
<br />
<strong><font size=5 id="Result">Result</font></strong><BR><BR><br />
coming soon…<BR><BR><br />
<br />
<img src=""><BR><br />
Fig.9 Comparison of normalized the GFP fluorescence intensity to cell density (FI/OD<SUB>600</SUB>).<BR><BR><br />
<strong><font size=5 id="Discussion">Discussion</font></strong><BR><BR><BR><BR><BR><br />
<br />
<p align=center><strong><font size=6 id="Future work"><ins>Future work</ins></font></strong></p><BR><BR><br />
<br />
<p style="line-height:110%"> <br />
We are going to construct the HHR fused with RNA scaffold (HHR-RNA scaffold) and no self-cleavage mutant (HHR*-RNA scaffold). HHR-RNA scaffold is unable to form RNA scaffold without self-cleavage because RNA scaffold contain complementary sequence to HHR. So we will evaluate whether the formation of RNA scaffold is regulated by self-cleavage or not. After that, we will construct RNA oscillation system based on HHR that periodically release an RNA fragment containing RNA scaffold and result in engineering "Twinkle. coli". We will try to make Split luciferase and fuse the split luciferase to MS2 and PP7. By using the Split luciferase, <I>E. coli</I> emit light periodically like a firefly. <BR><br />
The oscillation system can also use its RNA scaffold for enzyme reactions. We sought other aptamers to increase the variety of BioBrick for enzyme reactions. Hung-Wei Yiu has reported RNA detection in living bacterial cells caused by binding of two RNA binding peptide, HTLV-1 Rex peptide and λN peptide, to two RNA aptamer. They fused these two peptides to split GFP and detect RNA which contains HTLV-1 Rex peptide and λN peptide aptamer. So we will construct and evaluate the system and resister as a BioBrick. <br />
</P><br />
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<BR><br />
<BR><br />
<BR><br />
<BR><br />
<strong>Reference</strong><BR><br />
<br />
<p style="line-height:110%"> <br />
[1] Natalia E. Broude, “Analysis of RNA localization and metabolism in single live bacterial cells: achievements and challenges” Molecular Microbiology (2011) 80(5), 1137-1147<BR><BR><br />
<br />
[2]Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye, F.A., “Organization of Intracellular Reactions with Rationally Designed RNA Assemblies” Science 333, 470-474 (2011).<BR><BR><br />
<br />
[3] Hung-Wei Yiu, Vadim V. Demidov, Paul Toran, Charles R. Cantor and Natalia E. Broude, "RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides" Pharmaceuticals 2011, 4, 494-508<BR><BR><BR><BR><BR><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen
Team:Tokyo-NoKoGen
2013-09-28T03:10:32Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<h1 id="index_title"></h1><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=7>Introduction</font></p></font><br />
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<hr><br />
<hr size="3" width="(60%)" align="left"noshade><br />
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<p style="text-indent:2em"><font size=5><br />
We all have a biological clock which controls the periodicity of</p>many physiological functions such as blood pressure, body</p>temperature and</p> concentration of hormones. The systems that generate circadian rhythms</p> are called oscillator. Oscillator has been researched fordeepening our</p> knowledge of circadian rhythms, understanding genetic network and signal</p> transfer.</p><br />
<BR><br />
<br />
<div style="text-align:center"><br />
<img src="https://static.igem.org/mediawiki/2013/0/0d/Circadian_rhythm.PNG", height="300", width="450",alt=""><br />
</div><br />
<BR><br />
<div style="text-align:center"><font size=3><br />
Fig.1 Circadian rhythm</font><br />
</div><br />
<p style="text-indent:1em"><br />
Oscillator is expected to be used for various applications.</p><br />
<BR><br />
<p style="text-indent:1em"><br />
Therefore, desired features of oscillator are:</p><br />
<p style="text-indent:4em"><br />
<strong><br />
1. Variation in types</strong></p><br />
<p style="text-indent:4em"><br />
<strong><br />
2. Feasibility in designing and controlling</strong></p><br />
<BR><br />
<p style="text-indent:2em"><br />
However, existing protein oscillator does not satisfy the above requirements.</p><br />
<img src="https://static.igem.org/mediawiki/2013/9/91/Protein.PNG",height="200", width="200"alt="" style="float:right"><br />
<p style="text-indent:2em"><br />
The first difficulty of protein oscillator is the small type of the protein. Because the number of repressor proteins and promoters are limited, the variation of protein oscillator is not many. </p><br />
<p style="text-indent:2em"><br />
Another difficulty of protein oscillator is the component, the protein. Protein requires several steps; transcription and translation, protein modification, folding, and degradation. When we want to construct or control such protein oscillator, we have to genetically engineer to change the transcriptional efficiency, translational efficiency and degradation ability. Therefore, it is difficult to design such gene circuits that can express the repressors periodically. </p><br />
<BR><br />
<p style="text-indent:4em"><font size=3><br />
Fig. 2 Steps of protein expression</font></p><br />
<BR><br />
<p style="text-indent:2em"><br />
To satisfy the requirements, we propose the New type of oscillator circuit – <span style="color:#ff0000">RNA oscillator</span>.</p><br />
<br />
<p style="text-indent:2em"><br />
Whereas there are only a few types of proteins that can be used for the construction of protein oscillator, many kinds of oscillators can be constructed with RNA by changing a few base. Therefore, it will allow us to construct a multiple and orthogonal oscillators.</p><br />
<p style="text-indent:2em"><br />
Another advantage is that, although protein has several steps until degradation, RNA has no steps of translation, modification and time-consuming degradation. Therefore, RNA oscillator can be designed and controlled by only changing the transcriptional efficiency and binding ability. It is easy to change the binding ability of RNA because all we have to do is to alter the base, and hence change the strength of base pairs binding.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/5/57/ProteinRNA.PNG",height="200",width="200",alt=""><br />
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<img src="https://static.igem.org/mediawiki/2013/6/61/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A3aa.PNG", height="300", width="350", alt="",style="float:right"><br />
<p style="text-indent:1em"><font size=3><br />
Fig.3 Steps of protein expression and RNA transcription Fig.4 RNA is easy to modify</font><br />
<BR><br />
<BR><br />
<p style="text-indent:2em"><br />
Like this, RNA meets the necessary standards for:</p><br />
<br />
<p style="text-indent:4em"><br />
<strong><br />
1. Variation in types</strong></p><br />
<p style="text-indent:4em"><br />
<strong><br />
2. Feasibility in designing and controlling</strong></p><br />
<BR><br />
<p style="text-indent:2em"><br />
Therefore, it can be used for combination with diverse mechanisms.<br />
For example, we can change output signals from RNA to other ones such as protein with reporter circuit, and control the oscillation with light sensor.</p><br />
<br />
<p style="text-indent:2em"><br />
In this way, the ability to combine with various mechanisms can be used for controlling complex systems such as drug delivery.</p><br />
<p style="text-indent:2em"><br />
We named the E. coli which has this ability Twinkle.coli because of its brilliant future.<br />
</p><br />
<div style="text-align:center"><br />
<img src="https://static.igem.org/mediawiki/2013/f/fa/Twinklecoli.PNG", height="300", width="350", alt=""><br />
<BR><br />
<BR><br />
<BR><br />
<div style="text-align:center"><font size=3><br />
Fig.5 Twinkle. coli</font><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice
Team:Tokyo-NoKoGen/Humanpractice
2013-09-28T03:07:53Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<p align=center><font size=3> <br />
When we talk about iGEM and synthetic biology, we talk about dealing with <i>E. coli</I> and <br />
<BR>DNA and all that... but hey! Isn't that dangerous? Don't you get infected from <i>E. coli</i>? <br />
<BR>Isn't genetic modification a bad thing? People might wonder.<br />
<BR> This page provides you an insight to how safe synthetic biology is, and how fun it can be. <br />
<BR>To let more people know, we thought that this should also be written in Japanese.<br />
<BR><br />
<BR><br />
<font size=2> 私たちは合成生物学の世界大会iGEMで大腸菌を用いて遺伝子組み換えを行っています。<br />
<BR>「え!大腸菌?大腸菌って感染するんじゃないの?」<br />
<BR>「お腹壊さない?」「遺伝子組み換えとかヤバイんじゃない??」<br />
<BR>・・・ハイ、よく言われます。<br />
<BR>そんな誤解を解くために、このページでは合成生物学の安全と、<br />
<BR>合成生物学の面白さを紹介したいと思います。一人でも多くの人に知ってもらうために、<br />
<br>このページは日本語でも紹介します!<BR><br />
<BR><br />
<BR> <br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety> How to work safely in the laboratory</a><br />
<BR><br />
<BR><font size=4>安全に実験するために</font><br />
<BR><br />
<BR> <br />
<BR><br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire> Questionnaire on Synthetic Biology</a><img src=https://static.igem.org/mediawiki/2013/1/11/Q7.JPG width=100><br />
<BR><br />
<BR><font size=4>合成生物学の意識調査</font><br />
<BR><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux>Do It Yourself</a></font><img src=https://static.igem.org/mediawiki/2013/3/39/Ikaika.jpg width=100><br />
<BR><font size=5>Ika Lux</font><br />
<br />
<BR><p align=center><iframe width="420" height="315" src="//www.youtube.com/embed/QoZZVeJH6IA" frameborder="0" allowfullscreen></iframe><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan>Team interactions</a></font><br />
<BR><br />
<BR><font size=4>他のチームとの交流</font><br />
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<li class="NK"><a target="_blank" href="http://www.tuat.ac.jp/en/index.html"><img class ="NK"src="https://static.igem.org/mediawiki/2013/7/76/東京農工大学.png"></a></li><br />
<li class="ST"><a target="_blank" href="http://www.tuat.ac.jp/~tanpaku/"><img src="https://static.igem.org/mediawiki/2013/5/5a/早出・津川研究室.png" ></a></li><br />
<li class="IK"><a target="_blank" href="http://www.tuat.ac.jp/~kakusan/index.html"><img src="https://static.igem.org/mediawiki/2013/8/87/池袋研究室.png"></a></li><br />
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</html></div>
Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice
Team:Tokyo-NoKoGen/Humanpractice
2013-09-28T03:03:46Z
<p>Miyake: </p>
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<p align=center><font size=3> <br />
When we talk about iGEM and synthetic biology, we talk about dealing with <i>E. coli</I> and <br />
<BR>DNA and all that... but hey! Isn't that dangerous? Don't you get infected from <i>E. coli</i>? <br />
<BR>Isn't genetic modification a bad thing? People might wonder.<br />
<BR> This page provides you an insight to how safe synthetic biology is, and how fun it can be. <br />
<BR>To let more people know, we thought that this should also be written in Japanese.<br />
<BR><br />
<BR><br />
<font size=2> 私たちは合成生物学の世界大会iGEMで大腸菌を用いて遺伝子組み換えを行っています。<br />
<BR>「え!大腸菌?大腸菌って感染するんじゃないの?」<br />
<BR>「お腹壊さない?」「遺伝子組み換えとかヤバイんじゃない??」<br />
<BR>・・・ハイ、よく言われます。<br />
<BR>そんな誤解を解くために、このページでは合成生物学の安全と、<br />
<BR>合成生物学の面白さを紹介したいと思います。一人でも多くの人に知ってもらうために、<br />
<br>このページは日本語でも紹介します!<BR><br />
<BR><br />
<BR> <br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety> How to work safely in the laboratory</a><br />
<BR><br />
<BR><font size=4>安全に実験するために</font><br />
<BR><br />
<BR> <br />
<BR><br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire> Questionnaire on Synthetic Biology</a><img src=https://static.igem.org/mediawiki/2013/1/11/Q7.JPG width=100><br />
<BR><br />
<BR><font size=4>合成生物学の意識調査</font><br />
<BR><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux>Do It Yourself</a></font><img src=https://static.igem.org/mediawiki/2013/3/39/Ikaika.jpg width=100><br />
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<BR><font size=4>Ika Lux</font><br />
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<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan>Team interactions</a></font><br />
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<BR><font size=4>他のチームとの交流</font><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice
Team:Tokyo-NoKoGen/Humanpractice
2013-09-28T03:00:45Z
<p>Miyake: </p>
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<a href="https://2013.igem.org"><img class="iGEM" src="https://static.igem.org/mediawiki/2013/5/58/IGEM.png"></a> <br />
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<td class="home"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen"><span class="home"></span></a></td><br />
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<BR><br />
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<p align=center><font size=3> <br />
When we talk about iGEM and synthetic biology, we talk about dealing with <i>E. coli</I> and <br />
<BR>DNA and all that... but hey! Isn't that dangerous? Don't you get infected from <i>E. coli</i>? <br />
<BR>Isn't genetic modification a bad thing? People might wonder.<br />
<BR> This page provides you an insight to how safe synthetic biology is, and how fun it can be. <br />
<BR>To let more people know, we thought that this should also be written in Japanese.<br />
<BR><br />
<BR><br />
<font size=2> 私たちは合成生物学の世界大会iGEMで大腸菌を用いて遺伝子組み換えを行っています。<br />
<BR>「え!大腸菌?大腸菌って感染するんじゃないの?」<br />
<BR>「お腹壊さない?」「遺伝子組み換えとかヤバイんじゃない??」<br />
<BR>・・・ハイ、よく言われます。<br />
<BR>そんな誤解を解くために、このページでは合成生物学の安全と、<br />
<BR>合成生物学の面白さを紹介したいと思います。一人でも多くの人に知ってもらうために、<br />
<br>このページは日本語でも紹介します!<BR><br />
<BR><br />
<BR> <br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety> How to work safely in the laboratory</a><br />
<BR><br />
<BR><font size=4>安全に実験するために</font><br />
<BR><br />
<BR> <br />
<BR><br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire> Questionnaire on Synthetic Biology</a><br />
<BR><br />
<BR><font size=4>合成生物学の意識調査</font><br />
<BR><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux>Do It Yourself</a></font><img src=https://static.igem.org/mediawiki/2013/3/39/Ikaika.jpg width=100><br />
<BR><br />
<BR><font size=4>Ika Lux</font><br />
<br />
<BR><p align=center><iframe width="420" height="315" src="//www.youtube.com/embed/QoZZVeJH6IA" frameborder="0" allowfullscreen></iframe><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan>Team interactions</a></font><br />
<BR><br />
<BR><font size=4>他のチームとの交流</font><br />
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<li class="NK"><a target="_blank" href="http://www.tuat.ac.jp/en/index.html"><img class ="NK"src="https://static.igem.org/mediawiki/2013/7/76/東京農工大学.png"></a></li><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice
Team:Tokyo-NoKoGen/Humanpractice
2013-09-28T02:59:35Z
<p>Miyake: </p>
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<BR><br />
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<p align=center><font size=3> <br />
When we talk about iGEM and synthetic biology, we talk about dealing with <i>E. coli</I> and <br />
<BR>DNA and all that... but hey! Isn't that dangerous? Don't you get infected from <i>E. coli</i>? <br />
<BR>Isn't genetic modification a bad thing? People might wonder.<br />
<BR> This page provides you an insight to how safe synthetic biology is, and how fun it can be. <br />
<BR>To let more people know, we thought that this should also be written in Japanese.<br />
<BR><br />
<BR><br />
<font size=2> 私たちは合成生物学の世界大会iGEMで大腸菌を用いて遺伝子組み換えを行っています。<br />
<BR>「え!大腸菌?大腸菌って感染するんじゃないの?」<br />
<BR>「お腹壊さない?」「遺伝子組み換えとかヤバイんじゃない??」<br />
<BR>・・・ハイ、よく言われます。<br />
<BR>そんな誤解を解くために、このページでは合成生物学の安全と、<br />
<BR>合成生物学の面白さを紹介したいと思います。一人でも多くの人に知ってもらうために、<br />
<br>このページは日本語でも紹介します!<BR><br />
<BR><br />
<BR> <br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety> How to work safely in the laboratory</a><br />
<BR><br />
<BR><font size=4>安全に実験するために</font><br />
<BR><br />
<BR> <br />
<BR><br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire> Questionnaire on Synthetic Biology</a><br />
<BR><br />
<BR><font size=4>合成生物学の意識調査</font><br />
<BR><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux>Do It Yourself</a></font><br />
<BR><br />
<BR><font size=4>Ika Lux</font><br />
<br />
<BR><p align=center><iframe width="420" height="315" src="//www.youtube.com/embed/QoZZVeJH6IA" frameborder="0" allowfullscreen></iframe><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan>Team interactions</a></font><br />
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<BR><font size=4>他のチームとの交流</font><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice
Team:Tokyo-NoKoGen/Humanpractice
2013-09-28T02:57:17Z
<p>Miyake: </p>
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<p align=center><font size=3> <br />
When we talk about iGEM and synthetic biology, we talk about dealing with <i>E. coli</I> and <br />
<BR>DNA and all that... but hey! Isn't that dangerous? Don't you get infected from <i>E. coli</i>? <br />
<BR>Isn't genetic modification a bad thing? People might wonder.<br />
<BR> This page provides you an insight to how safe synthetic biology is, and how fun it can be. <br />
<BR>To let more people know, we thought that this should also be written in Japanese.<br />
<BR><br />
<BR><br />
<font size=2> 私たちは合成生物学の世界大会iGEMで大腸菌を用いて遺伝子組み換えを行っています。<br />
<BR>「え!大腸菌?大腸菌って感染するんじゃないの?」<br />
<BR>「お腹壊さない?」「遺伝子組み換えとかヤバイんじゃない??」<br />
<BR>・・・ハイ、よく言われます。<br />
<BR>そんな誤解を解くために、このページでは合成生物学の安全と、<br />
<BR>合成生物学の面白さを紹介したいと思います。一人でも多くの人に知ってもらうために、<br />
<br>このページは日本語でも紹介します!<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<br />
<BR> <br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety> How to work safely in the laboratory</a><br />
<BR><br />
<BR><font size=4>安全に実験するために</font><br />
<BR><br />
<BR> <br />
<BR><br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire> Questionnaire on Synthetic Biology</a><br />
<BR><br />
<BR><font size=4>合成生物学の意識調査</font><br />
<BR><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux>Do It Yourself</a></font><br />
<BR><br />
<BR><font size=4>Ika Lux</font><br />
<br />
<BR><p align=center><iframe width="420" height="315" src="//www.youtube.com/embed/QoZZVeJH6IA" frameborder="0" allowfullscreen></iframe><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan>Team interactions</a></font><br />
<BR><br />
<BR><font size=4>他のチームとの交流</font><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire
Team:Tokyo-NoKoGen/questionnaire
2013-09-28T02:47:10Z
<p>Miyake: </p>
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<h1 id="index_title"></h1><br />
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<br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>Safety in the laboratoy</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on synthetic biology</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux"><li><strong>Do It Yourself -Ika Lux-</strong></li></a><br />
<br />
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<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=6>Questionnaire on synthetic biology</font></p><br />
<BR><br />
<BR><br />
<p align=center><font size=4>We would like to thank all the people who helped us answer the questionnaire, we were able to get 85 replies by 2 p.m. on 27th of September. We prepared 21 questions to find out how people recognize synthetic biology. </p></font><br />
<br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q1. Are you a male or a female?</font></p><br />
<p align=center><font size=3>性別を教えてください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/%E3%82%B9%E3%83%A9%E3%82%A4%E3%83%891.JPG></p><br />
<BR><br />
<BR><br />
<p align=center><font size=4>The ratio is almost ideal!</font></p><br />
<br />
<BR><br />
<p align=center><font size=4>Q2. What age are you?</font></p><br />
<p align=center><font size=3>年代を教えてください</p></font><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1f/Q2.JPG></p><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Unfortunately, the ratio of generations is biased. This means that this questionnaire tend to reflect younger people's opinion.</font></p><br />
<BR><br />
<p align=center><font size=4>Q3. Are you specialized in science or non-science?</font></p><br />
<p align=center><font size=3>文系か理系、どちらがお得意かお答えください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/2c/Q3.JPG></p><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Most of people are major in science. </font></p><br />
<BR><br />
<p align=center><font size=4>Q4. Do you know synthetic biology?</font></p><br />
<p align=center><font size=3>「合成生物学」を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/Q4.JPG></p><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Even though many people who involved in this questionnaire are major in science, it seems that the word "Synthetic biology" is unfamiliar.</font></p><br />
<BR><br />
<p align=center><font size=4>Q5. If yes, how did you get to know about it?</font></p><br />
<p align=center><font size=3> 「はい」と答えた方はどこで知りましたか?<br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/03/5.jpg></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q6. What do you think Synthetic biology is about?</font></p><br />
<p align=center><font size=3> 合成生物学は次のうちどのような学問だと思いますか?<br />
<p alignt=center><img src=https://static.igem.org/mediawiki/2013/f/f9/Q6.JPG width=600></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q7. Do you know iGEM?</font></p><br />
<p align=center><font size=3>iGEMという大会を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/11/Q7.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q8. If yes, how do you know it?</font></p><br />
<p align=center><font size=3>「はい」と答えた方はどこで知りましたか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/9/9f/Q8.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q9. How do you pronounce "iGEM"?</font></p><br />
<p align=center><font size=3>"iGEM"の読み方は次のうちどれだと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/04/Q9.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>10. Where do you think the iGEM Jamboree is held?</font></p><br />
<p align=center><font size=3>iGEMの本選はどこで開催されていると思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/19/Q10.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q11. What do you think is important in iGEM?</font></p><br />
<p align=center><font size=3>iGEMとはどのようなことを競う大会だと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q11.JPG width=800></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q12. How many Japanese teams do you think participate iGEM?</font></p><br />
<p align=center><font size=3>iGEMの日本のチームは何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e1/Q12.JPG></p><br />
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<p align=center><font size=4>Q13. How many teams do you think there are in the whole iGEM society?</font></p><br />
<p align=center><font size=3>世界にはiGEMが何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8f/Q13.JPG></P><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q14. What do you think of, when you hear "E. coli"?</font></p><br />
<p align=center><font size=3>「大腸菌」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/c/cf/Q14.JPG></P><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q15. What do you think of, when you hear "Genetic modification"?</font></p><br />
<p align=center><font size=3>「遺伝子組み換え」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q15.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q16. Did you know that genetically modified E. coli can collect heavy metal ions?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を使って重金属を回収することが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/a/a0/Q16.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q17. Did you know that genetically modified E. coli can become luminescent?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌が青色に光ることが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/40/Q17.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q18. Did you know that genetically modified E. coli can be regulated by light?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を光で制御出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/22/Q18.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q19. We bought a squid at a nearby supermarket. What do you think we did with them?</font></p><br />
<p align=center><font size=3>私達は近所のスーパーで石川県産のイカを買いました。?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e6/Q19.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q20. After this questionnaire, do you think synthetic biology is interesting?</font></p><br />
<p align=center><font size=3>合成生物学について、なんとなく興味を持っていただけたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/d/db/Q20.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q21. Do you also feel that you want to participate iGEM competitiong?</font></p><br />
<p align=center><font size=3>iGEMに参加したいと思ってもらえたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/5a/Q21.JPG></p><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire
Team:Tokyo-NoKoGen/questionnaire
2013-09-28T02:46:11Z
<p>Miyake: </p>
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<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>Safety in the laboratoy</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on synthetic biology</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux"><li><strong>Do It Yourself -Ika Lux-</strong></li></a><br />
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<p align=center><font size=6>Questionnaire on synthetic biology</font></p><br />
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<p align=center><font size=4>We would like to thank all the people who helped us answer the questionnaire, we were able to get 85 replies by 2 p.m. on 27th of September. We prepared 21 questions to find out how people recognize synthetic biology. </p></font><br />
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<p align=center><font size=4>Q1. Are you a male or a female?</font></p><br />
<p align=center><font size=3>性別を教えてください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/%E3%82%B9%E3%83%A9%E3%82%A4%E3%83%891.JPG></p><br />
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<p align=center><font size=4>The ratio is almost ideal!</font></p><br />
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<p align=center><font size=4>Q2. What age are you?</font></p><br />
<p align=center><font size=3>年代を教えてください</p></font><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1f/Q2.JPG></p><br />
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<p align=center><font size=4>Unfortunately, the ratio of generations is biased. This means that this questionnaire tend to reflect younger people's opinion.</font></p><br />
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<p align=center><font size=4>Q3. Are you specialized in science or non-science?</font></p><br />
<p align=center><font size=3>文系か理系、どちらがお得意かお答えください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/2c/Q3.JPG></p><br />
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<p align=center><font size=4>Most of people are major in science. </font></p><br />
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<p align=center><font size=4>Q4. Do you know synthetic biology?</font></p><br />
<p align=center><font size=3>「合成生物学」を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/Q4.JPG></p><br />
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<p align=center><font size=4>Even though many people who involved in this questionnaire are major in science, it seems that the word "Synthetic biology" is unfamiliar.</font></p><br />
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<p align=center><font size=4>Q5. If yes, how did you get to know about it?</font></p><br />
<p align=center><font size=3> 「はい」と答えた方はどこで知りましたか?<br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/03/5.jpg></p><br />
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<p align=center><font size=4>Q6. What do you think Synthetic biology is about?</font></p><br />
<p align=center><font size=3> 合成生物学は次のうちどのような学問だと思いますか?<br />
<p alignt=center><img src=https://static.igem.org/mediawiki/2013/f/f9/Q6.JPG width=600></p><br />
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<p align=center><font size=4>Q7. Do you know iGEM?</font></p><br />
<p align=center><font size=3>iGEMという大会を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/11/Q7.JPG></p><br />
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<p align=center><font size=4>Q8. If yes, how do you know it?</font></p><br />
<p align=center><font size=3>「はい」と答えた方はどこで知りましたか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/9/9f/Q8.JPG></p><br />
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<p align=center><font size=4>Q9. How do you pronounce "iGEM"?</font></p><br />
<p align=center><font size=3>"iGEM"の読み方は次のうちどれだと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/04/Q9.JPG></p><br />
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<p align=center><font size=4>10. Where do you think the iGEM Jamboree is held?</font></p><br />
<p align=center><font size=3>iGEMの本選はどこで開催されていると思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/19/Q10.JPG></p><br />
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<p align=center><font size=4>Q11. What do you think is important in iGEM?</font></p><br />
<p align=center><font size=3>iGEMとはどのようなことを競う大会だと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q11.JPG></p><br />
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<p align=center><font size=4>Q12. How many Japanese teams do you think participate iGEM?</font></p><br />
<p align=center><font size=3>iGEMの日本のチームは何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e1/Q12.JPG></p><br />
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<p align=center><font size=4>Q13. How many teams do you think there are in the whole iGEM society?</font></p><br />
<p align=center><font size=3>世界にはiGEMが何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8f/Q13.JPG></P><br />
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<p align=center><font size=4>Q14. What do you think of, when you hear "E. coli"?</font></p><br />
<p align=center><font size=3>「大腸菌」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/c/cf/Q14.JPG></P><br />
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<p align=center><font size=4>Q15. What do you think of, when you hear "Genetic modification"?</font></p><br />
<p align=center><font size=3>「遺伝子組み換え」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q15.JPG></p><br />
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<p align=center><font size=4>Q16. Did you know that genetically modified E. coli can collect heavy metal ions?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を使って重金属を回収することが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/a/a0/Q16.JPG></p><br />
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<p align=center><font size=4>Q17. Did you know that genetically modified E. coli can become luminescent?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌が青色に光ることが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/40/Q17.JPG></p><br />
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<p align=center><font size=4>Q18. Did you know that genetically modified E. coli can be regulated by light?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を光で制御出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/22/Q18.JPG></p><br />
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<p align=center><font size=4>Q19. We bought a squid at a nearby supermarket. What do you think we did with them?</font></p><br />
<p align=center><font size=3>私達は近所のスーパーで石川県産のイカを買いました。?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e6/Q19.JPG></p><br />
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<p align=center><font size=4>Q20. After this questionnaire, do you think synthetic biology is interesting?</font></p><br />
<p align=center><font size=3>合成生物学について、なんとなく興味を持っていただけたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/d/db/Q20.JPG></p><br />
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<p align=center><font size=4>Q21. Do you also feel that you want to participate iGEM competitiong?</font></p><br />
<p align=center><font size=3>iGEMに参加したいと思ってもらえたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/5a/Q21.JPG></p><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire
Team:Tokyo-NoKoGen/questionnaire
2013-09-28T02:45:25Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>Safety in the laboratoy</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on synthetic biology</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux"><li><strong>Do It Yourself -Ika Lux-</strong></li></a><br />
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<p align=center><font size=6>Questionnaire on synthetic biology</font></p><br />
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<p align=center><font size=4>We would like to thank all the people who helped us answer the questionnaire, we were able to get 85 replies by 2 p.m. on 27th of September. We prepared 21 questions to find out how people recognize synthetic biology. </p></font><br />
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<p align=center><font size=4>Q1. Are you a male or a female?</font></p><br />
<p align=center><font size=3>性別を教えてください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/%E3%82%B9%E3%83%A9%E3%82%A4%E3%83%891.JPG></p><br />
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<p align=center><font size=4>The ratio is almost ideal!</font></p><br />
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<p align=center><font size=4>Q2. What age are you?</font></p><br />
<p align=center><font size=3>年代を教えてください</p></font><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1f/Q2.JPG></p><br />
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<p align=center><font size=4>Unfortunately, the ratio of generations is biased. This means that this questionnaire tend to reflect younger people's opinion.</font></p><br />
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<p align=center><font size=4>Q3. Are you specialized in science or non-science?</font></p><br />
<p align=center><font size=3>文系か理系、どちらがお得意かお答えください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/2c/Q3.JPG></p><br />
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<p align=center><font size=4>Most of people are major in science. </font></p><br />
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<p align=center><font size=4>Q4. Do you know synthetic biology?</font></p><br />
<p align=center><font size=3>「合成生物学」を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/Q4.JPG></p><br />
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<p align=center><font size=4>Even though many people who involved in this questionnaire are major in science, it seems that the word "Synthetic biology" is unfamiliar.</font></p><br />
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<p align=center><font size=4>Q5. If yes, how did you get to know about it?</font></p><br />
<p align=center><font size=3> 「はい」と答えた方はどこで知りましたか?<br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/03/5.jpg></p><br />
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<p align=center><font size=4>Q6. What do you think Synthetic biology is about?</font></p><br />
<p align=center><font size=3> 合成生物学は次のうちどのような学問だと思いますか?<br />
<p alignt=center><img src=https://static.igem.org/mediawiki/2013/f/f9/Q6.JPG></p><br />
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<p align=center><font size=4>Q7. Do you know iGEM?</font></p><br />
<p align=center><font size=3>iGEMという大会を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/11/Q7.JPG></p><br />
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<p align=center><font size=4>Q8. If yes, how do you know it?</font></p><br />
<p align=center><font size=3>「はい」と答えた方はどこで知りましたか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/9/9f/Q8.JPG></p><br />
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<p align=center><font size=4>Q9. How do you pronounce "iGEM"?</font></p><br />
<p align=center><font size=3>"iGEM"の読み方は次のうちどれだと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/04/Q9.JPG></p><br />
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<p align=center><font size=4>10. Where do you think the iGEM Jamboree is held?</font></p><br />
<p align=center><font size=3>iGEMの本選はどこで開催されていると思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/19/Q10.JPG></p><br />
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<p align=center><font size=4>Q11. What do you think is important in iGEM?</font></p><br />
<p align=center><font size=3>iGEMとはどのようなことを競う大会だと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q11.JPG></p><br />
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<BR><br />
<p align=center><font size=4>Q12. How many Japanese teams do you think participate iGEM?</font></p><br />
<p align=center><font size=3>iGEMの日本のチームは何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e1/Q12.JPG></p><br />
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<p align=center><font size=4>Q13. How many teams do you think there are in the whole iGEM society?</font></p><br />
<p align=center><font size=3>世界にはiGEMが何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8f/Q13.JPG></P><br />
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<p align=center><font size=4>Q14. What do you think of, when you hear "E. coli"?</font></p><br />
<p align=center><font size=3>「大腸菌」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/c/cf/Q14.JPG></P><br />
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<p align=center><font size=4>Q15. What do you think of, when you hear "Genetic modification"?</font></p><br />
<p align=center><font size=3>「遺伝子組み換え」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q15.JPG></p><br />
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<p align=center><font size=4>Q16. Did you know that genetically modified E. coli can collect heavy metal ions?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を使って重金属を回収することが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/a/a0/Q16.JPG></p><br />
<BR><br />
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<BR><br />
<p align=center><font size=4>Q17. Did you know that genetically modified E. coli can become luminescent?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌が青色に光ることが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/40/Q17.JPG></p><br />
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<p align=center><font size=4>Q18. Did you know that genetically modified E. coli can be regulated by light?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を光で制御出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/22/Q18.JPG></p><br />
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<p align=center><font size=4>Q19. We bought a squid at a nearby supermarket. What do you think we did with them?</font></p><br />
<p align=center><font size=3>私達は近所のスーパーで石川県産のイカを買いました。?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e6/Q19.JPG></p><br />
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<p align=center><font size=4>Q20. After this questionnaire, do you think synthetic biology is interesting?</font></p><br />
<p align=center><font size=3>合成生物学について、なんとなく興味を持っていただけたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/d/db/Q20.JPG></p><br />
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<p align=center><font size=4>Q21. Do you also feel that you want to participate iGEM competitiong?</font></p><br />
<p align=center><font size=3>iGEMに参加したいと思ってもらえたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/5a/Q21.JPG></p><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire
Team:Tokyo-NoKoGen/questionnaire
2013-09-28T02:44:43Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<h1 id="index_title"></h1><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>Safety in the laboratoy</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on synthetic biology</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux"><li><strong>Do It Yourself -Ika Lux-</strong></li></a><br />
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<p align=center><font size=6>Questionnaire on synthetic biology</font></p><br />
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<p align=center><font size=4>We would like to thank all the people who helped us answer the questionnaire, we were able to get 85 replies by 2 p.m. on 27th of September. We prepared 21 questions to find out how people recognize synthetic biology. </p></font><br />
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<p align=center><font size=4>Q1. Are you a male or a female?</font></p><br />
<p align=center><font size=3>性別を教えてください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/%E3%82%B9%E3%83%A9%E3%82%A4%E3%83%891.JPG></p><br />
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<p align=center><font size=4>The ratio is almost ideal!</font></p><br />
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<p align=center><font size=4>Q2. What age are you?</font></p><br />
<p align=center><font size=3>年代を教えてください</p></font><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1f/Q2.JPG></p><br />
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<p align=center><font size=4>Unfortunately, the ratio of generations is biased. This means that this questionnaire tend to reflect younger people's opinion.</font></p><br />
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<p align=center><font size=4>Q3. Are you specialized in science or non-science?</font></p><br />
<p align=center><font size=3>文系か理系、どちらがお得意かお答えください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/2c/Q3.JPG></p><br />
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<p align=center><font size=4>Most of people are major in science. </font></p><br />
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<p align=center><font size=4>Q4. Do you know synthetic biology?</font></p><br />
<p align=center><font size=3>「合成生物学」を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/Q4.JPG></p><br />
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<p align=center><font size=4>Even though many people who involved in this questionnaire are major in science, it seems that the word "Synthetic biology" is unfamiliar.</font></p><br />
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<p align=center><font size=4>Q5. If yes, how did you get to know about it?</font></p><br />
<p align=center><font size=3> 「はい」と答えた方はどこで知りましたか?<br />
<p align=center><https://static.igem.org/mediawiki/2013/0/03/5.jpg></p><br />
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<p align=center><font size=4>Q6. What do you think Synthetic biology is about?</font></p><br />
<p align=center><font size=3> 合成生物学は次のうちどのような学問だと思いますか?<br />
<p alignt=center><img src=https://static.igem.org/mediawiki/2013/f/f9/Q6.JPG></p><br />
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<p align=center><font size=4>Q7. Do you know iGEM?</font></p><br />
<p align=center><font size=3>iGEMという大会を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/11/Q7.JPG></p><br />
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<p align=center><font size=4>Q8. If yes, how do you know it?</font></p><br />
<p align=center><font size=3>「はい」と答えた方はどこで知りましたか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/9/9f/Q8.JPG></p><br />
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<p align=center><font size=4>Q9. How do you pronounce "iGEM"?</font></p><br />
<p align=center><font size=3>"iGEM"の読み方は次のうちどれだと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/04/Q9.JPG></p><br />
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<p align=center><font size=4>10. Where do you think the iGEM Jamboree is held?</font></p><br />
<p align=center><font size=3>iGEMの本選はどこで開催されていると思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/19/Q10.JPG></p><br />
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<BR><br />
<p align=center><font size=4>Q11. What do you think is important in iGEM?</font></p><br />
<p align=center><font size=3>iGEMとはどのようなことを競う大会だと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q11.JPG></p><br />
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<p align=center><font size=4>Q12. How many Japanese teams do you think participate iGEM?</font></p><br />
<p align=center><font size=3>iGEMの日本のチームは何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e1/Q12.JPG></p><br />
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<p align=center><font size=4>Q13. How many teams do you think there are in the whole iGEM society?</font></p><br />
<p align=center><font size=3>世界にはiGEMが何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8f/Q13.JPG></P><br />
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<p align=center><font size=4>Q14. What do you think of, when you hear "E. coli"?</font></p><br />
<p align=center><font size=3>「大腸菌」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/c/cf/Q14.JPG></P><br />
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<p align=center><font size=4>Q15. What do you think of, when you hear "Genetic modification"?</font></p><br />
<p align=center><font size=3>「遺伝子組み換え」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q15.JPG></p><br />
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<p align=center><font size=4>Q16. Did you know that genetically modified E. coli can collect heavy metal ions?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を使って重金属を回収することが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/a/a0/Q16.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q17. Did you know that genetically modified E. coli can become luminescent?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌が青色に光ることが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/40/Q17.JPG></p><br />
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<p align=center><font size=4>Q18. Did you know that genetically modified E. coli can be regulated by light?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を光で制御出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/22/Q18.JPG></p><br />
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<p align=center><font size=4>Q19. We bought a squid at a nearby supermarket. What do you think we did with them?</font></p><br />
<p align=center><font size=3>私達は近所のスーパーで石川県産のイカを買いました。?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e6/Q19.JPG></p><br />
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<p align=center><font size=4>Q20. After this questionnaire, do you think synthetic biology is interesting?</font></p><br />
<p align=center><font size=3>合成生物学について、なんとなく興味を持っていただけたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/d/db/Q20.JPG></p><br />
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<p align=center><font size=4>Q21. Do you also feel that you want to participate iGEM competitiong?</font></p><br />
<p align=center><font size=3>iGEMに参加したいと思ってもらえたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/5a/Q21.JPG></p><br />
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Miyake
http://2013.igem.org/File:5.jpg
File:5.jpg
2013-09-28T02:43:06Z
<p>Miyake: uploaded a new version of &quot;File:5.jpg&quot;</p>
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/light
Team:Tokyo-NoKoGen/light
2013-09-28T02:40:03Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<li><a href="#Introduction"><strong>Introduction</strong></a></li><br />
<li><a href="#Objective"><strong>Objective</strong></a></li><br />
<li><a href="#Method"><strong>Method</strong></a><br />
<ul><br />
<li><a href="#-Design">-Design</a></li><br />
<li><a href="#-Parts construction">-Parts construction</a></li><br />
</ul><br />
</li><br />
<li><a href="#-Evaluation"><strong>-Evaluation</strong></a><br />
<ul><br />
<li><a href="#Result">Result</a></li><br />
</ul><br />
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<li><a href="#Future work"><strong>Future work</strong></a></li><br />
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<font size=5><br />
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<BR><br />
<p align=center><font size=7><strong>Light sensor</strong></font></p><br />
<BR><br />
<BR><br />
<BR> <br />
<font size=6 id="Introduction"><strong>Introduction</strong></font><br />
<BR><br />
<BR><br />
<BR><br />
<h3>1. YF1/ FixJ</h3><br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
YF1/ FixJ system is a blue light (480 nm) sensing system. YF1 is a fusion protein, heme-binding PAS sensor domain of FixL from <I>Bradyrhizobium japonicum</I>(FixL) and the LOV blue light sensor domain of <I>Bacillus subtilis</I> YtvA(YtvA). The Histidine kinase YF1 employs a light-oxygen-voltage, blue light photosensor domain. FixJ is YF1’s cognate response regulator. In the absence of blue light, YF1 phosphorylates FixJ, and phosphorylated FixJ drives robust gene expression from the FixK2 promoter(Ref. 1,2,3).<br />
</p><br />
<br />
<BR><BR><br />
<img src=https://static.igem.org/mediawiki/2013/8/88/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A316.PNG><br />
<br />
<BR><BR><br />
<br />
<h3>2. Rhodopsin</h3><br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
Halophilic archaea, such as <I>Halobacterium salinarum</I> and<I> Natronobacterium pharaonis (N. pharaonis)</I> show phototaxis by responding to changes in light color and intensity using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. signals to Htr proteins via helix-helix interaction. Htr protein consists of two transmembrane helices and a cytoplasmic methyl-accepting and His-Kinase domain, and belongs to histidine kinase / phosphoreregulator two-component system for regulating cells’ flagellar motors for phototaxis (Ref. 4,5).<br />
</p><br />
<BR><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/39/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A317.PNG><br />
<br />
<BR><BR><br />
<br />
<BR><p style="line-height:110%"><br />
<font size=6 id="Objective"><strong>Objective</strong></font><br />
<br />
<BR><BR><BR><br />
<br />
Reguration of taRNA expression by light sensor protein<BR><br />
</p><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6 id="Method"><strong>Method</strong></font><br />
<br />
<BR><BR><BR><br />
<br />
<font size=5 id="-Design"><strong>-Design</strong></font><br />
<br />
<BR><br />
<p style="line-height:110%"><br />
We construct HHRs containing RBS downstream of the P<sub>const</sub> promoter (low), and taRNA which binds HHR and inactivates HHR’s self-cleaving activity is placed downstream of P<sub>ompC</sub> or P<sub>fixk2</sub> promoter. Under dark condition, taRNA is expressed and inactivates HHR’s self-cleaving activity. On the other hand, under blue light condition, taRNA isn’t expressed and HHR self-cleaves. Becouse of HHR self-cleaving, GFP’s RBS is exposed and GFP is expressed. <BR><br />
</p><br />
<BR><br />
<BR><br />
<font size=5 id="-Parts construction"><strong>-Parts construction</strong></font><br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
1. YF1/ FixJ<br />
<br />
<BR><BR><br />
<br />
1.) BioBrick part BBa_K1053210 (Tokyo-NoKoGen2013) is fused with HHR connecting with GFP (BBa_K1053004(Tokyo-NoKoGen2013)) or HHR* connecting with GFP (BBa_K1053005(Tokyo-NoKoGen2013)) by using Overlap PCR. <br />
<br />
<BR><BR><br />
<br />
2.) The PCR products were gel purified and digested with <I>Xba</I>Ⅰ and <I>Pst</I>Ⅰ. The digested products were ligated into pSB1A3 vector.(Fig. 1)<BR><BR><br />
<br />
3.) Constructed plasmids were transformed into <I>E.coli</I> DH5α.<br />
</P><br />
<br />
<BR><BR><BR><br />
<br />
<img src=https://static.igem.org/mediawiki/2013/2/29/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A33.PNG><br />
<BR>Fig. 1 pSB1A3 – P<sub>const.</sub> – YF1/FixJ - P<sub>fixk2</sub> – TR(12)- HHR - GFP - DT<BR><BR><BR><BR><br />
<img src=https://static.igem.org/mediawiki/2013/5/51/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A34.PNG><BR><br />
Fig. 2 pSB1A3- P<sub>const.</sub> – YF1/FixJ - P<sub>fixk2</sub> – TR(12)- HHR - GFP - DT<br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
2. Rhodopsin<br />
<br />
<BR><BR><br />
<br />
1.) BioBrick part BBa_K769003 (Tokyo-NoKoGen2012) which consists of a chimeric sensory rhodopsin and its cognate transducer from <I>N. pharaonis</I> and the histidine kinase domain of EnvZ from <I>E. coli</I>, that is fused with HHR connecting with GFP (BBa_K1053004(Tokyo-NoKoGen2013)) or HHR* connecting with GFP (BBa_K1053005(Tokyo-NoKoGen2013)) by using Overlap PCR. <br />
<br />
<BR><BR><br />
<br />
2.) The PCR products were gel purified and digested with <I>Eco</I>RⅠ and <I>Pst</I>Ⅰ. The digested products were ligated into pSB1A3 vector.(Fig. 3)<br />
<br />
<BR><BR><br />
<br />
3.) Constructed plasmids were transformed into <I>E.coli</I> DH5α.<br />
<br />
<BR><BR><BR><BR><br />
<br />
<img src=https://static.igem.org/mediawiki/2013/e/e0/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A37.PNG><BR><br />
Fig.3 pSB1A3 - P<sub>const.</sub> - SRⅡ- HtrⅡ- EnvZ - P<sub>ompC</sub> - HHR - GFP - DT<BR><BR><BR><BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/35/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A38.PNG><BR><br />
Fig.4 pSB1A3 - P<sub>const.</sub> - SRⅡ- HtrⅡ- EnvZ - P<sub>ompC</sub> - HHR* - GFP - DT<BR><BR><BR><BR><br />
</p><br />
<br />
<br />
<BR><br />
<BR><br />
<font size=6 id="-Evaluation"><strong>-Evaluation</strong></font><br />
<br />
<BR><BR><BR><BR><br />
<br />
<p style="line-height:110%"><br />
YF1/ FixJ<BR><br />
1.) Construct made in –Parts construction- was used to transform into <I>E.coli</I> DH5α.<br />
<br />
<BR><BR><BR><br />
<br />
2.) The transformants were pre-cultured in 3 mL LB medium overnight at 37 degrees celsius, under dark condition.<br />
<br />
<BR><BR><BR><br />
<br />
3.) 450 μL of pre-cultures were inoculated into 3 mL LB medium inside and incubated either under dark or blue light conditions. OD595 and GFP fluorescence intensity were measured at certain time.(Fig. 1)<BR><BR><BR><br />
4.) We evaluated P<sub>const.</sub> – taR12- P<sub>const.</sub> (low) – YF1/ FixJ – P<sub>fixk2</sub> – HHR(Fig. 6).<BR><BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/37/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A313.PNG><BR><br />
Fig. 6 P<sub>const.</sub> – taR12- P<sub>const.</sub> (low) – YF1/ FixJ – P<sub>fixk2</sub> - HHR<BR><BR><br />
<p style="line-height:110%"><br />
We estimate in light GFP is expressed by HHR’s self cleavage and in dark GFP isn’t expressed by taRNA’s expression. If HHR’s active site is mutated, GFP isn’t expressed.<br />
The GFP fluorescence intensity was taken after main culture for 12 h by Plate Reader in this evaluation method.<BR><br />
</p><br />
<br />
<br />
</p><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/e/e8/Parts.jpg><br />
<BR><BR><BR><br />
<font size=5 id="Result"><strong>Result</strong></font><br />
<BR><BR><br />
<p style="line-height:110%"><br />
There is no clear difference of light and dark condition of HHR-GFPuv or HHR*-GFPuv.<BR><br />
The cause of this result is that excitation light of GFP and YF1 is almost the same.<BR><br />
</p><br />
<img src=https://static.igem.org/mediawiki/2013/b/b5/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A39.PNG><br />
<br />
<BR><BR><br />
<br />
Result 1 Evaluation of transformed <I>E.coli</I> under the light or dark condition.<br />
<BR><BR><br />
<BR><br />
<p style="line-height:110%"><br />
If HHR’s active site isn’t mutated difference between in light or dark is observed. This result is suggested that in light taRNA is expressed and HHR’s auto cleavage is inhibited.<BR><br />
RNA oscillator’s result is difference between taRNA’s existence or absence isn’t observed. We think that this difference is caused by difference between P<sub>const.</sub>(low) and P<sub>bad.</sub><BR><br />
</P><br />
<br />
<img src=https://static.igem.org/mediawiki/2013/c/c8/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A310.PNG><br />
<br />
<BR><BR><br />
<br />
Result 2 Evaluation of transformed <I>E. coli</I> under the light or dark condition.<br />
<BR><BR><BR><br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="Future work"><strong>Future work</strong></font><br />
<BR><br />
<p style="line-height:110%"><br />
We want to evaluate P<sub>const.</sub> - Yf1/FixJ - P<sub>fixk2</sub>- TR(12)- HHR- GFP- DT and P<sub>fixk2</sub> - taR12 - P<sub>const.</sub>(low) TR(12)- HHR- GFP- P<sub>const.</sub>- YF1/ FixJ by changing main culture time and changing evaluation method again.<BR><br />
We want to synchronize oscillation cycle with <I>Twinkle.coli</I> by light sensor protein in the future.<BR><br />
</p><br />
<BR><br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
Reference<BR><br />
[1] <I>J. Mol. Biol.</I>(2009) 385, 1433-1444<BR><br />
[2] <I>J. Mol. Biol. </I>(2012) 416, 534-542<BR><br />
[3] <I>Bacteriology </I>(1998) 180, 5251-5255<BR><br />
[4] Xue-Nong Zhang <I>et al.</I> (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <BR><br />
[5] Wouter D. Hoff <I>et al.</I> (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct.<BR><br />
<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Diary
Team:Tokyo-NoKoGen/Diary
2013-09-28T02:37:11Z
<p>Miyake: </p>
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<h1 id="index_title"></h1><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Diary"><li><strong>Diary<strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Protocol"><li><strong>Protocol<strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Safety"><li><strong>Safety</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Acknowledgement"><li><strong>Acknowledgement</strong></li><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Attribution"><li><strong>Attribution</strong></a></li><br />
<br />
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<BR><br />
<h1 id="april">April</h1><br />
<BR><strong>18</strong><br />
<p><br />
Our first official gathering of Tokyo-NoKoGen 2013! Okay, let’s deicde on what we will do for the project this year.<br />
<BR><br />
<BR><strong>25</strong><br />
<p>Before we start, we looked up on what projects have been in presented in the past iGEM competitions. <br />
Each one of us picked a team of our interest, and made a presentation to the team.<br />
<BR><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/35/Line.jpg><br />
<br />
<br />
<h1 id="may">May</h1><br />
<BR><strong>1</strong><br />
<BR>To understand how iGEM work, we made a seminar to understand the basics, for example how to use restriction enzymes, and how to use the BioBrick parts.<br />
<BR><br />
<BR><strong>16</strong><br />
<BR>Since we are going to present at Tokyo University very very soon, we need to practice and revise our poster presentation...Are we all ready?<br />
<BR><BR><strong>19</strong><br />
<BR>School festival at Tokyo University! We made a poster presentation to many high school students and parents on our last year's project. It was very interesting to explain about genetic engineering of E. coli to those who are not familiar with them! We also got to know other team members of iGEM Japan, they were people who were fun to be with.<br />
<BR><br />
<BR><strong>25</strong><br />
<BR>Homework for the next iGEM meeting - each person must look up on a recent paper and make a presentation to the team.<br />
<br />
<br />
<BR><br />
<br />
<img src=https://static.igem.org/mediawiki/2013/3/35/Line.jpg><br />
<h1 id="june">June</h1><br />
<BR><strong>6</strong><br />
<BR>Brainstorming...production of biodiesel? Make fertilisers using E. coli?<br />
<BR><br />
<BR><strong>20</strong><br />
<BR>Another brainstorming...shall we make an extended version of lux luminescence? Anyone with good idea? Since we are running out of time, we wil make it a compulsory for each person to come up with at least 3 ideas!<br />
<BR><br />
<br />
<br />
<img src=https://static.igem.org/mediawiki/2013/3/35/Line.jpg><br />
<h1 id="july">July</h1><br />
<strong>4</strong><br />
<BR>Everyone came up with many interesting ideas over the past two weeks. Hmmm....which one shall we challenge? Not so much time left.<br />
<BR><br />
<BR><strong>11</strong><br />
<BR>Brainstorming...Ooo, HHR, RNA oscillator...?<br />
<BR><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/35/Line.jpg><br />
<h1 id="august">August</h1><br />
<BR><strong>14</strong><br />
<BR>Yay, we got BBa_K1053000, our first ribozyme!<br />
<BR><br />
<BR><strong>18</strong><br />
<BR>Ohki: E-mail was sent to me(Ohki). It said as follows: Oscillator group members are Ito, Sakamoto and Takabayashi (modeling). This was the first time I had to make a simulation for RNA oscillation. Hey, who decided on that?<br />
<BR><br />
<BR><strong>19</strong><br />
<BR>Ohki: I(Ohki) decided the plan roughly. First of all, I researched what kind of software there are. I also studied how they simulate the entire system, in the protein oscillation article. <br />
<BR><br />
<BR><strong>22</strong><br />
<BR>Akiko, Nana:Plac-RBS, Ptet-RBS, PcI-RBS, tetRllite-DT, λCI-lite-DT, lacI-lite-DT was constructed <br />
<BR><br />
<BR><strong>25</strong><br />
<BR>iGEM Japan Party (this time, I(Ohki) didnt simulate at all)<br />
<BR><br />
<BR><strong>27</strong><br />
<BR>Ohki: I installed the cellillustrator. This is the software which enables us to simulate biological phenomenon. But, in this software, I was not able to simulate concisely. For example, I was not able to consider the generation of complex between HHR and taRNA. Because I roughly simulated the system, the oscillation occured. This was an obvious result because it was set to be so.<br />
<BR><br />
<BR><strong>28</strong><br />
<BR>We dicided on wiki, poster and oral presentation members<br />
<BR>Ohki: Prof. Sode said to me, "parameter in this model was not enough at all." So, I decided to recreate the simulation and decided that I was going to use other software.<br />
<BR><br />
<BR><strong>30</strong><br />
<BR>Ippei, Shunsuke: We got BBa_K1053003 and BBa_K1053004<br />
<BR>Akiko, Nana: We confirmed sequences<br />
<br />
<br />
<br />
<img src=https://static.igem.org/mediawiki/2013/3/35/Line.jpg><br />
<br />
<br />
<BR><br />
<BR><br />
<h1 id="september">September</h1><br />
<BR><strong>3</strong><br />
<BR>Ippei, Shunsuke: Made TOP10 competent cells<br />
<BR>Akiko, Nana: constructed pSB1C3-Plac-RVS-tetR-lite-DT and pSB1C3-PcI-RBS-lacI-lite-DT<br />
<BR>Akiko, Nana:We confirmed sequences of pSB1C3- Plac -RBS -tetR -lite –-DT (BBa_K1053301), pSB1C3- PcI -RBS -lacI -lite –DT (BBa_K1053302).<br />
<BR><br />
<BR><strong>5</strong><br />
<BR>Eri, Masaki: Construction of RNA scaffold (containing MS2/PP7 aptamer) BBa_K1053110 <br />
<BR>Akiko, Nana: We constructed pSB1C3- Ptet -RBS -λcI -lite –DT.<br />
<BR><br />
<BR><strong>7</strong><br />
<BR>Eri, Masaki:Sequence analysis of constructed "RNA scaffold" parts. Okay, the sequence seems fine!<br />
<BR><br />
<BR><strong>9</strong><br />
<BR>Ippei, Shunsuke: Construction of pSB3C5-PBAD-HHR-GRP-DT and evaluation. Hopefully it works!<br />
<BR>Akiko, Nana: We confirmed the sequence of PcI -RBS and pSB1C3- Ptet -RBS -cI -lite -DT(BBa_K1053300).<br />
<BR><br />
<BR><strong>10</strong><br />
<BR>Get ready for BioBrick submition! Woohoo<br />
<BR>Eri, Masaki: We received our synthesized genes, FA-linker-MS2 and FB-linker-PP7! Yes! <br />
<BR><br />
<BR><strong>11</strong><br />
<BR>Eri, Masaki: The three separate fragments were ligated together, however obtained no colonies. We decided to construct using another method.<br />
<BR>Akiko, Nana: We constructed pSB1c3-Plac-RBS-tetR-lite-DT-Pcl-RBS-lacI-lite-DT.<br />
<BR><br />
<BR><strong>12</strong><br />
<BR>Ohki: I installed Simbiology and studied how this software works.<br />
<BR>Akiko, Nana: We constructed pSB1c3-Plac-RBS-tetR-lite-DT-Pcl-RBS-lacI-lite-DT-Ptet-RBS-λcI-lite-DT .<br />
<BR><br />
<BR><strong>13</strong><br />
<BR>Ippei, Shunsuke: RE-evaluation of pSB3C5-PBAD-HHR-GRP-DT and evaluation. Hopefully it works this time.<br />
<BR><br />
<BR><strong>16</strong><br />
<BR>Ippei, Shunsuke: We got three HHRs composing RNA oscillator and HHR fused with RNA scaffold. But oh no...they have extra <i>Xba</I>I :(<br />
<BR>Akiko, Nana: We constructed pSB1A2-PtetO-1-GFPaav-DT, pSB1A2-PtetO-1-GFPlva-DT, however we couldn’t confirm its sequences.<br />
<BR><br />
<BR><strong>19</strong><br />
<BR>Ohki: I simulated our RNA oscillator and noticed this system doesn't work by substituting any parameters.<br />
<BR><br />
<BR><strong>20</strong><br />
<BR>Ippei, Shunsuke: Evaluation of pSB3C5-PBad-tr(12)-HHR-GFP-DT by taking a time course. When is the optimal time to see the difference?<br />
<BR>Eri: Time to order our T-shirt!<br />
<BR><img src=https://static.igem.org/mediawiki/2013/a/aa/%EF%BC%B4%E3%82%B7%E3%83%A3~1.PNG width=300><br />
<BR>Ohki: I formularized differential equation to confirm whether oscillation doesn't occur and solve them.<br />
<BR><br />
<BR><strong>22</strong><br />
<BR>Eri, Masaki: Construction of split GFP fused with MS2/PP7 proteins, Pconst(w)-RBS-MS2-RBS-PP7-DT<br />
<BR><br />
<BR><strong>23</strong><br />
<BR>Eri, Masaki: Evaluated GFP fluorescence in <i>E. coli</i> expressing split GFP fused with proteins and RNA scaffold. But we detected no fluorescence....why?<br />
<BR>Yukimi, Chihiro: Evalution of light sensors. Seems to behave differently under light and under dark!<br />
<BR>Akiko, Nana: We confirmed the sequence of pSB1c3-Plac-RBS-tetR-lite-DT-Pcl-RBS-lacI-lite-DT-Ptet-RBS-λcI-lite-DT, but we found it had a different sequence from other source. We decided to stop this experiment and we will achieve to construct and evaluate the protein oscillator by MOT.<br />
<BR><br />
<BR><strong>26</strong><br />
<BR>Evaluation of pSB1A3-PBAD-tr(42)-HHR-taR(12)-DT-Pconst(H)-taR(42)-DT<br />
<BR>Ohki: Time to finish writing the wiki.<br />
<BR><br />
<BR><strong>27</strong><br />
<BR>Wiki FREEZE<br />
<BR><br />
<BR><br />
<BR><img src=https://static.igem.org/mediawiki/2013/3/35/Line.jpg><br />
<h1 id="october">October</h1><br />
<BR><strong>5</strong><br />
<BR><font size=7 color=red>Asia Regional Jamboree</font><br />
<BR><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/8/81/DSC01616.JPG width=800><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/modeling
Team:Tokyo-NoKoGen/modeling
2013-09-28T02:23:34Z
<p>Miyake: </p>
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<ul id="contents"><br />
<a href="#Background"><li><strong>Background</strong></li></a><br />
<a href="#Method"><li><strong>Method</strong></li></a><br />
<a href="#Results and discussion"><li><strong>Results and discussion</strong></li></a><br />
<a href="#Reference"><li><strong>Reference</strong></li></a><br />
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<p align=center><font size=7>Modeling</font></p></font><br />
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<p align=center><font size=6 id="Background"><strong><span style="border-bottom:double 6px #0000ff;">Background</span></font></strong></p><br />
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<p style="line-height:110%">We constructed oscillation circuit using <font color="#ff0000">RNA</font>. We think that it is easier to design the system than protein oscillation, and protein oscillator is difficult to control because there are many process such like translation and activation of related proteins. So, it can be used as a very useful tool which has a lot of applications. In our assumption, RNA oscillation using <font color="#ff0000">h</font>ammer<font color="#ff0000">h</font>ead <font color="#ff0000">r</font>ibozyme(HHR) is suitable for our system. But does this system really generate the oscillation? Or, if so, what is the ideal conditions of generation of the oscillation and how much faster than our constructed system is than protein oscillator? To investigate this, we created simple model using Simbiology.</p><br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/11/Oscillation_circuit.PNG></p><br />
<p align=center>Fig1. The scheme of RNA oscillation using Simbiology.</p><br />
<p><br />
<p style="line-height:110%">Simbiology is Math Works softare, MATLAB’s extension. This software allows us to simulate biological phenomenon of the species, reactions, and compartments that make up the system (in our system, component of RNA oscillation, such like, HHRs, and taRNAs). For this model, we simulated that how condition this system works (Fig 1).</p><br />
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<p align=center><font size=6 id="Method"><strong><span style="border-bottom:double 6px #0000ff;">Method</span></strong></font></p><br />
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Before simulation of our construction of RNA oscillator, we estimated the values below:<br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/0f/Table1a.PNG width=700></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/a/ab/Table2a.PNG width=700 height=500></p><br />
<BR><br />
We used these values to simulate our RNA oscillation.<br />
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<p align=center><font size=6 id="Results and discussion"><strong><span style="border-bottom:double 6px #0000ff;">Results and discussion</span></strong></font></p><br />
<BR><br />
We used the Simbiology for the simulation, and the result(<em>K<sub>&#945;</sub></em>=0.005, <em>K<sub>on</sub></em> = 2.0, <em>K<sub>off</sub></em> = 1.0) is shown as the graph below.<br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/86/A%3D0.005%2Ckon%3D0.01%2Ckoff%3D1.0.PNG></p><br />
<p align=center>Fig2. Time scale of relative amount of HHR1(<font color="#0000ff">blue line</font>), taRNA1(<font color="#008000">green line)</font> and HHR1 and taRNA1 complex(<font color="#ff0000">red line</font>).</p><br />
<p style="line-height:110%"> Results from simulation show that the amount of HHR-taRNA complex, HHR and taRNA reached steady state(X axis means Time(sec), and Y axis means molecules of each species). So, the result shows that this system doesn't generate oscillation. But we substituted false values into <em>K<sub>on</sub></em> and <em>K<sub>off</sub></em>. So, to confirm whether oscillation doesn't occur any time, we formularized differential equations as follows:</p><br />
<BR><br />
<dl><dd><img src=https://static.igem.org/mediawiki/2013/6/68/Formula1.PNG></dd></dl><br />
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<p style="line-height:110%">where the <em>K<sub>d</sub></em> is the rate of RNA degradation, <em>K<sub>&#945;</sub></em> is rate of self-cleavage frequency, the constant <em>K<sub>cons</sub></em> is the rate of production of new RNA, and [・] is the concentration of "・".(taR[i]HR[i+1] means taRNAi and HRRi+1 complex(i=1,2,3))</p><br />
<BR><br />
<p style="line-height:110%">To generate oscillation, we focused on the steady state. If oscillation occurs, we’ll get recurrence formula from above differential equations and it shows bi-steady state exists, which is feature of generating oscillation. </p><br />
<BR><br />
<p style="line-height:110%">In steady state, the changes of each species are 0. Therefore, the following equations are satisfied.</p><br />
<BR><br />
<dl><dd><img src=https://static.igem.org/mediawiki/2013/4/4f/Equation_value1.PNG></dd></dl><br />
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And, we get these values immediately as follows,<br />
<BR><br />
<dl><dd><img src=https://static.igem.org/mediawiki/2013/b/b3/Formula3.PNG></dd></dl><br />
<BR><br />
<p style="line-height:110%">From the results (4)~(6) shows that concentration of all of the associated species go to the constant value in steady state, which means that there isn't bi-steady state and that this system doesn't generate the oscillation. When we began this project, we assumed that taRNA inhibits the self-cleavage of HHR and causes a decrease in the amount of HHR. So the oscillation should have occurred. But even when the taRNA inhibits HHR’s activity, HHR itself is continuously expressed and new HHRs come one after another , which leads the concentration of HHR and taRNA to the constant value which depend on <em>K<sub>on</sub></em>, <em>K<sub>off</sub></em>s. <em>K<sub>cons</sub></em>,<em>K<sub>&#945;</sub></em>and <em>K<sub>d</sub></em>. To generate oscillation in our system, we should have introduced the factor which stops transcription directly, such like protein repressor and attenuater.</p><br />
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<p align=center><font size=6 id="Reference"><strong><span style="border-bottom:double 6px #0000ff;">Reference</span></strong></font></p><br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
<sup>i</sup> Bnenedikt Klauser and Jorg S. Hartig, (2013), An engineered small RNA-mediated genetic switch based on a ribozyme expression platform, Nucleic Acid Res. ,41(10), 5542-5552<br />
<BR><br />
<sup>ii</sup> Bremer.H., Dennis.P.P(1996) Modulation of chemical composition and other parameters of the cell by growth rate.<br />
<BR><br />
<sup>iii</sup> Gerardo.F., James M. Smith, Robert Cedergren(1998), Schistosome Satellite DNA Encodes Active Hammerhead Ribozymes. Mol Cell Biol. 18(7), 3880-3888<br />
<BR><br />
<sup>iv</sup> Bruce Alberts, <em>et.al</em>, MOLECULAR BIOLOGY OF THE CELL 4th edition (New York, Newton Press, 2004 )<br />
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<td class="home"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen"><span class="home"></span></a></td><br />
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<div id="index"><br />
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<h1 id="index_title"></h1><br />
<ul id="contents"><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>Safety in the laboratoy</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on synthetic biology</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux"><li><strong>Do It Yourself -Ika Lux-</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan"><li><strong>iGEM Japan</strong></li></a><br />
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<div id="main"><br />
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<p align=center><font size=6>Team interactions</font></p><br />
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<BR>1. Gathering of iGEM teams in Japan<br />
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<BR>2. Helping our friend Sumbawagen in creating their wiki</font><br />
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<p align=center><font size=6 color=green>Gathering of iGEM teams in JAPAN</font></p><br />
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This year, 11 teams from Japan are participating the iGEM competition:Biwako_Nagahama, Osaka, Kyoto, Tokyo_Tech, UT-Tokyo, Chiba, KIT-Kyoto, TMU-Tokyo, HokkaidoU_Japan, KAIT_Kapan, Tokyo-NoKoGen. Since we have many teams within the country, it is a pity we do not use this opportunity to get together and have a discussion on each other's project and if possible, make collaboration to make an even better project! So this year, we participated at a festival in May held at Tokyo University and an event in August held at Tokyo Metropolitan University.<br />
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日本から今年、11のチームがiGEMに参加しています:Biwako_Nagahama, Osaka, Kyoto, Tokyo_Tech, UT-Tokyo, Chiba, KIT-Kyoto, TMU-Tokyo, HokkaidoU_Japan, KAIT_Kapan, Tokyo-NoKoGenです。こんなに多くのチームが日本国内にいるのに、集まらないでいるのはとてももったいないことです。私達は今年も、5月には東京大学の五月祭でポスター発表を、8月には東京首都大学で開催された交流会に参加しました。その時の様子を写真でお見せします。<br />
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<img src=https://static.igem.org/mediawiki/2013/9/94/DSC00599.JPG width=400><br />
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<p align=center><font size=6 color=green>Helping our friend Sumbawagen on their wiki</font></p><br />
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<BR>Dr. Arief Budi Witarto <br />
Julmansyah, the team instructor of Sumbawagen 2013 team, has been our good friend working in the same laboratory for many years. He is a very gentl and kind man, who we enjoyed working with. This year, he went back to his homeland Indonesia, where he has become a Dean of Technobiology Faculty ofSumbawa University of Technology and started an iGEM team with the 1st year students at his new university.<br />
About a few weeks ago, our instructor Prof. Sode visited their team and made his stay a very present time. Prof. Sode came bakc to Japan telling us how impressive Sumbawagen's laboratory work has been, however, he also told us that they were having problems uploading data on their wiki. It did not take us a long time to decide on helping them, so we immediately took contact with their team to start the cooperation. If you have time, please visit Sumbawagen team's wiki, which can be accessed from the link below:<br />
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<img src=https://static.igem.org/mediawiki/2013/9/99/Sumbawagen.jpg width=800><br />
<BR><p align=center><a href=https://2013.igem.org/Team:Sumbawagen>https://2013.igem.org/Team:Sumbawagen</a></p></br><br />
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http://youtu.be/QoZZVeJH6IA<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/ikalux
Team:Tokyo-NoKoGen/ikalux
2013-09-28T00:55:32Z
<p>Miyake: </p>
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<h1 id="index_title">Human practice</h1><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>How to work safely in the laboratory</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on Synthetic Biology</strong></li></a><br />
<font color="orange"><li><strong>Do It Yourself -IkaLux-</strong></li></font><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan"><li><strong>Team interactions: iGEM JAPAN</strong></li></a><br />
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<p align=center><img src="https://static.igem.org/mediawiki/2013/3/39/Ikaika.jpg"></p><br />
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<p align=center><font size=6>DO IT YOURSELF</font></p><br />
<p align=center><font size=5>Simple Experiment to do at home</font></p><br />
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<BR><br />
<p align=center><font size=4> Please have a look at our video first</font></p><br />
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<BR><p align=center><iframe width="420" height="315" src="//www.youtube.com/embed/QoZZVeJH6IA" frameborder="0" allowfullscreen></iframe></p><br />
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<font size=4>We hope you enjoyed our video! The video shows you how it is easy to culture a luminescent bacteria at home, a very simple experiment with only a need to buy a fresh squid from a nearby supermarket. The boy and a girl shown in the video are 1st year students at our university, who have no experience in doing laboratory experiments. Even they can do it for the first time - why don't you also try?</font><br />
<BR><br />
<font size=4>ビデオは楽しんでいただけたでしょうか?このビデオでは、光るバクテリアを家で簡単に培養出来るということ、スーパーで新鮮なイカを買ってくるだけで出来てしまうということを伝えたく、作りました。ビデオに登場する男の子と女の子は大学1年生で、まだ複雑な実験を経験したことがありません。あなたもぜひ家で培養してみませんか?</font><br />
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<p align=center><font size=6> Steps in preparing ika culture</p></font><br />
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<font size=4>As shown in the video, the steps needed for making a squid is very simple. The photos below will help you remember how the steps were taken. First you buy a fresh ika, then take the inside away and cut it into an appropriate size. Put that piece into a container filled with 3% NaCl. Leave this ika for 1 to 2 weeks at 4℃, you will soon start to see luminescent "spots" on the surface of ika - luminescent bacteria!An agarose gel can be easily made from boiling the rest of ika for 3 to 5 minutes, and mix it well with agar.</font><br />
<BR><br />
<font size=4>ビデオでもお見せした通り、イカを用意するのはとても簡単です。下の写真は、ビデオでお見せした内容の復習です。まず、近所のスーパーで新鮮なイカを買ってきます。そのイカをちょうど良い大きさに切りだします。切り出した断片を3% NaClに浸し、4℃で1週間から2週間程度培養します。すると、イカの表面に光る”斑点”が見えてきますが、これが発光バクテリアです!寒天培地もとても簡単に用意できます。余ったイカをお鍋で3分から5分にて、寒天と混ぜるだけです。<br />
<BR><img src=https://static.igem.org/mediawiki/2013/5/5a/I01.jpg width=300><br />
<img src=https://static.igem.org/mediawiki/2013/d/de/I02.jpg width=300><br />
<img src=https://static.igem.org/mediawiki/2013/b/b7/I03.jpg width=300><br />
<img src=https://static.igem.org/mediawiki/2013/e/ef/I04.jpg width=300><br />
<img src=https://static.igem.org/mediawiki/2013/3/3c/I05.jpg width=300><br />
<img src=https://static.igem.org/mediawiki/2013/8/8f/I06.jpg width=300><br />
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<p align=center><font size=6> Obtain the luminescent bacteria</p></font><br />
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<BR>We were not satisfied with only finding the luminescent bacteria, but we also wanted to know what it is. Therefore we decided to culture the colony on the agar plate to get a single colony. Can you see it?<br />
<BR>私達は発光バクテリアを見つけるだけでは満足が出来ませんでした。そこで、バクテリアの正体を探るために、寒天培地でバクテリアを培養してみました。見えるかな?<br />
<img src=https://static.igem.org/mediawiki/2013/8/86/Lumi.jpg><br />
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<BR><br />
<p align=center><font size=6> Cloning the bacterium gene</p></font><br />
<BR><br />
<BR><br />
<BR>For cloning the bacterium, we designed the following primer. We ran PCR and confirmed the bands by gel electrophoresis. The cloning successfully worked!<br />
<BR>バクテリアの遺伝子をクローニングするために、次のプライマーを設計しました。PCRを行ってバンドを電気泳動で確認したらバンドを得ることが出来ました。<br />
<img src=https://static.igem.org/mediawiki/2013/e/e7/Primers.jpg><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/f/f4/Questionnaire.jpg><br />
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<p align=center><font size=6> Sequence the cloned gene</p></font><br />
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<BR>We hurried and sequenced the cloned gene fragment. The sequencing data is shown below:<br />
<BR>バンドが見えたので私達は慌ててシーケンス解析を行いました。その結果が次の通りです:<br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/5/55/Sequence.jpg><br />
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<BR><br />
<BR>Using the obtained sequence, we made a blast analysis against a gene database (http://blast.ncbi.nlm.nih.gov/) to find out what bacterium we obtained on the surface of ika. As shown in the image below, we found that the gene is derived from Photobacterium Phosphoreum!<br />
<BR>得られた遺伝子が一体なんのバクテリア由来であるのかを調べるために、データベース(http://blast.ncbi.nlm.nih.gov/)に照らし合わせてみました。以下の図がその結果になりますが、Photobacterium Phosphoreumのバクテリアを得ることに成功しました!<br />
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<img src=https://static.igem.org/mediawiki/2013/4/45/Blast.jpg><br />
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<BR>When we hear the work "bacteria" or "cloning" it might scare some people away because it sounds very difficult. But as you can see, they are very very simple and are an experiment anyone can do. We hope that you wil also challenge yourself in culturing an ika lux!<br />
<BR>このように、「バクテリア」や「クローニング」と聞くととても難しいことのように思えますが、実はとても簡単です。だれでも出来るシンプルな実験です。あなたもぜひ一度ika luxを培養してみませんか?<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/labosafety
Team:Tokyo-NoKoGen/labosafety
2013-09-28T00:54:58Z
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>Safety in the laboratoy</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on synthetic biology</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux"><li><strong>Do It Yourself -Ika Lux-</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan"><li><strong>iGEM Japan</strong></li></a><br />
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<p align=center><font size=6>Safety in the laboratory</font><br />
<BR><BR><br />
<font size=4>実験室での安全を守るために</font></p><br />
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<hr><br />
<br />
<font size=3><br />
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<BR>We treat different kinds of bacteria in the laboratory. We also treat many toxic chemicals, which an do us serious harm if we are not careful enough. We shuold never forget the all possible accidents that can happen to us in the laboratory, and if something does happen, anything small for example electric cables were near the tap water, you should let all the lab members know so that we do not face any bigger problem.<br />
To avoid any problems, we should take good care of ourselves in the laboratory, and here below are a few tips on doing experiment.<br />
<BR><br />
私達は研究室で様々な菌体を扱い、また、毒性の高い試薬も扱います。交通事故と同じように、研究室でもどこに事故となりうる種があるか、常に私達はアンテナを張っていなければなりません。もし何か、どんなに小さなことでも、たとえば電気コンセントの近くに水たまりが出来ていたとか、研究室のメンバー全員に知らせ注意を呼びかけることが事故を防ぐために必要なことなのです。<br />
大きな事故を避けるためにも、普段から十分注意を払うことが、とても大事なのです。<br />
<p align=center><br />
<br />
<BR><br />
<BR>1. Put on gloves, lab coat and safety goggles. Always keep the benches clean that you don't spill anything dangerous.<br />
<BR><img src="https://static.igem.org/mediawiki/2013/8/86/P01.jpg" width=400><br />
<BR><br />
<BR><br />
<BR>2. These plastic bags contains garbage that are contaminated by <i>E. coli</I>. What we do is we autoclave them before throwing them away so that we do not spread any genetically modified bacteria. <br />
<img src="https://static.igem.org/mediawiki/2013/0/07/P02.jpg" width=400><br />
<BR><br />
<BR><br />
<BR>3. We also autoclave all equipment that we used for treating <i>E. coli</I>. After autoclaving, we put test tubes in the oven at 180 degrees celcius for 2 hours. This way, we make sure that all equipment we use over and over again are kept clean.<br />
<BR><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/P03.jpg" width=400><br />
<BR><br />
<BR><br />
<BR>4. What he is holding is a plate of colonies of <i>E. coli</i>. This experiment is done in a P2 level room, we do not leak any <i>E. coli</i> outside the room.<br />
<BR><img src="https://static.igem.org/mediawiki/2013/a/a0/P04.jpg" width=400><br />
<BR><br />
<BR><br />
<BR>5. We treat <i>E> coli</i> inside the clean bench. Before we tough them, we make sure that our hands are clean (although we have gloves on), we spray our hands with 70% ethanol.<br />
<img src="https://static.igem.org/mediawiki/2013/d/db/P05.jpg" width=400><br />
<BR><br />
<BR><br />
<BR>6. The clean bench looks like this. We look through the glass window when treating <i>E. coli</i> on agar plates or inside test tubes.<br />
<img src="https://static.igem.org/mediawiki/2013/0/03/P07.jpg width=400"><br />
<BR><br />
<BR><br />
<BR><br />
7. This is Eri with her growing <i>E. coli</i>. She looks very happy to find her bacteria growing steadily.Let people know that you are doing an experiment, so that you are not left forgotten. If something happens, someone should be aware and available to help you.<br />
<img src="https://static.igem.org/mediawiki/2013/5/5a/P08.jpg" width=400><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire
Team:Tokyo-NoKoGen/questionnaire
2013-09-28T00:54:16Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<h1 id="index_title"></h1><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety"><li><strong>Safety in the laboratoy</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire"><li><strong>Questionnaire on synthetic biology</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux"><li><strong>Do It Yourself -Ika Lux-</strong></li></a><br />
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<p align=center><font size=6>Questionnaire on synthetic biology</font></p><br />
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<p align=center><font size=4>We would like to thank all the people who helped us answer the questionnaire, we were able to get 85 replies by 2 p.m. on 27th of September. We prepared 21 questions to find out how people recognize synthetic biology. </p></font><br />
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<p align=center><font size=4>Q1. Are you a male or a female?</font></p><br />
<p align=center><font size=3>性別を教えてください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/%E3%82%B9%E3%83%A9%E3%82%A4%E3%83%891.JPG></p><br />
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<p align=center><font size=4>The ratio is almost ideal!</font></p><br />
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<BR><br />
<p align=center><font size=4>Q2. What age are you?</font></p><br />
<p align=center><font size=3>年代を教えてください</p></font><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/1f/Q2.JPG></p><br />
<BR><br />
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<p align=center><font size=4>Unfortunately, the ratio of generations is biased. This means that this questionnaire tend to reflect younger people's opinion.</font></p><br />
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<p align=center><font size=4>Q3. Are you specialized in science or non-science?</font></p><br />
<p align=center><font size=3>文系か理系、どちらがお得意かお答えください</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/2c/Q3.JPG></p><br />
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<p align=center><font size=4>Most of people are major in science. </font></p><br />
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<p align=center><font size=4>Q4. Do you know synthetic biology?</font></p><br />
<p align=center><font size=3>「合成生物学」を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/52/Q4.JPG></p><br />
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<p align=center><font size=4>Even though many people who involved in this questionnaire are major in science, it seems that the word "Synthetic biology" is unfamiliar.</font></p><br />
<BR><br />
<p align=center><font size=4>Q5. If yes, how did you get to know about it?</font></p><br />
<p align=center><font size=3> 「はい」と答えた方はどこで知りましたか?<br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/80/Q5.JPG></p><br />
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<p align=center><font size=4>Q6. What do you think Synthetic biology is about?</font></p><br />
<p align=center><font size=3> 合成生物学は次のうちどのような学問だと思いますか?<br />
<p alignt=center><img src=https://static.igem.org/mediawiki/2013/f/f9/Q6.JPG></p><br />
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<p align=center><font size=4>Q7. Do you know iGEM?</font></p><br />
<p align=center><font size=3>iGEMという大会を知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/11/Q7.JPG></p><br />
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<p align=center><font size=4>Q8. If yes, how do you know it?</font></p><br />
<p align=center><font size=3>「はい」と答えた方はどこで知りましたか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/9/9f/Q8.JPG></p><br />
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<p align=center><font size=4>Q9. How do you pronounce "iGEM"?</font></p><br />
<p align=center><font size=3>"iGEM"の読み方は次のうちどれだと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/0/04/Q9.JPG></p><br />
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<BR><br />
<p align=center><font size=4>10. Where do you think the iGEM Jamboree is held?</font></p><br />
<p align=center><font size=3>iGEMの本選はどこで開催されていると思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/1/19/Q10.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q11. What do you think is important in iGEM?</font></p><br />
<p align=center><font size=3>iGEMとはどのようなことを競う大会だと思いますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q11.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q12. How many Japanese teams do you think participate iGEM?</font></p><br />
<p align=center><font size=3>iGEMの日本のチームは何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e1/Q12.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q13. How many teams do you think there are in the whole iGEM society?</font></p><br />
<p align=center><font size=3>世界にはiGEMが何チームいるか知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8f/Q13.JPG></P><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q14. What do you think of, when you hear "E. coli"?</font></p><br />
<p align=center><font size=3>「大腸菌」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/c/cf/Q14.JPG></P><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q15. What do you think of, when you hear "Genetic modification"?</font></p><br />
<p align=center><font size=3>「遺伝子組み換え」という単語から連想するものは何ですか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/4b/Q15.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q16. Did you know that genetically modified E. coli can collect heavy metal ions?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を使って重金属を回収することが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/a/a0/Q16.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q17. Did you know that genetically modified E. coli can become luminescent?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌が青色に光ることが出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/4/40/Q17.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q18. Did you know that genetically modified E. coli can be regulated by light?</font></p><br />
<p align=center><font size=3>遺伝子組み換え型大腸菌を光で制御出来ることを知っていますか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/2/22/Q18.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q19. We bought a squid at a nearby supermarket. What do you think we did with them?</font></p><br />
<p align=center><font size=3>私達は近所のスーパーで石川県産のイカを買いました。?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e6/Q19.JPG></p><br />
<BR><br />
<BR><br />
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<p align=center><font size=4>Q20. After this questionnaire, do you think synthetic biology is interesting?</font></p><br />
<p align=center><font size=3>合成生物学について、なんとなく興味を持っていただけたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/d/db/Q20.JPG></p><br />
<BR><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Q21. Do you also feel that you want to participate iGEM competitiong?</font></p><br />
<p align=center><font size=3>iGEMに参加したいと思ってもらえたでしょうか?</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/5/5a/Q21.JPG></p><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice
Team:Tokyo-NoKoGen/Humanpractice
2013-09-28T00:53:09Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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</head><br />
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<div id="wrapper"><br />
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<div id="title_logo"> <br />
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<a href="https://2013.igem.org"><img class="iGEM" src="https://static.igem.org/mediawiki/2013/5/58/IGEM.png"></a> <br />
</div><br />
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<div id="mymenubar"><br />
<br />
<table><br />
<tbody><br />
<tr><br />
<td class="home"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen"><span class="home"></span></a></td><br />
<br />
<td class="project"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Project"><span class="project"></span></a></td><br />
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<td class="humanpractice1"><div class="humanpractice1"></div></td> <br />
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<td class="achievement"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Achievement"><span class="achievement"></span></a></td><br />
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<div id="main"><br />
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<BR><br />
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<br />
<p align=center><font size=3> <br />
When we talk about iGEM and synthetic biology, we talk about dealing with <i>E. coli</I> and <br />
<BR>DNA and all that... but hey! Isn't that dangerous? Don't you get infected from <i>E. coli</i>? <br />
<BR>Isn't genetic modification a bad thing? People might wonder.<br />
<BR> This page provides you an insight to how safe synthetic biology is, and how fun it can be. <br />
<BR>To let more people know, we thought that this should also be written in Japanese.<br />
<BR><br />
<BR><br />
<font size=2> 私たちは合成生物学の世界大会iGEMで大腸菌を用いて遺伝子組み換えを行っています。<br />
<BR>「え!大腸菌?大腸菌って感染するんじゃないの?」<br />
<BR>「お腹壊さない?」「遺伝子組み換えとかヤバイんじゃない??」<br />
<BR>・・・ハイ、よく言われます。<br />
<BR>そんな誤解を解くために、このページでは合成生物学の安全と、<br />
<BR>合成生物学の面白さを紹介したいと思います。一人でも多くの人に知ってもらうために、<br />
<br>このページは日本語でも紹介します!<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<br />
<BR> <br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/labosafety> How to work safely in the laboratory</a><br />
<BR><br />
<BR><font size=4>安全に実験するために</font><br />
<BR><br />
<BR> <br />
<BR><br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire> Questionnaire on Synthetic Biology</a><br />
<BR><br />
<BR><font size=4>合成生物学の意識調査</font><br />
<BR><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ikalux>Do It Yourself</a></font><br />
<BR><br />
<BR><font size=4>Ika Lux</font><br />
<BR><br />
<BR> <br />
<BR><br />
<font size=6><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/ijapan>Team interactions</a></font><br />
<BR><br />
<BR><font size=4>他のチームとの交流</font><br />
<br />
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<div id="space"><br />
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<br />
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<li class="UI"><a target="_blank" href="http://www.ultizyme.jp/"><img class="UI" src="https://static.igem.org/mediawiki/2013/4/47/アルティザイム・インターナショナル.jpg" ></a></li><br />
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<li class="LN"><a target="_blank" href="http://lne.st/"><img class="LN" src="https://static.igem.org/mediawiki/2013/2/2a/リバネス.png"></a></li><br />
<li class="IR"><a target="_blank" href="http://www.ikedarika.co.jp/english/"><img class="IR" src="https://static.igem.org/mediawiki/2013/b/b8/池田理化.gif"></a></li><br />
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<br />
<li class="NK"><a target="_blank" href="http://www.tuat.ac.jp/en/index.html"><img class ="NK"src="https://static.igem.org/mediawiki/2013/7/76/東京農工大学.png"></a></li><br />
<li class="ST"><a target="_blank" href="http://www.tuat.ac.jp/~tanpaku/"><img src="https://static.igem.org/mediawiki/2013/5/5a/早出・津川研究室.png" ></a></li><br />
<li class="IK"><a target="_blank" href="http://www.tuat.ac.jp/~kakusan/index.html"><img src="https://static.igem.org/mediawiki/2013/8/87/池袋研究室.png"></a></li><br />
</div><br />
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</div><br />
<br />
</body><br />
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</html></div>
Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:52:35Z
<p>Miyake: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg width=500></p><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=600></p><br />
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<p align=center><br />
Fig.5 Comparison of normalized GFPuv fluorescence under white light condition and dark condition.<br />
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Sensory rhodopsin : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1C3-<i>Pconst.-SRII-HtrII-EnvZ-PompC-GFP</i><br />
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Negative control : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1A3-<i>PompC-GFP</i><br />
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Positive control : <i>E.coli</i> BL21(DE3)/pSB1A3-<i>PompC-GFP</i> </p><br />
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<BR>As you can see from the result, we obtained a sensory rhodopsin fused by EnvZ that enhanced the down stream expression in the dark and repressed expression under light. For this year's project, we wanted to combine this light sensor with our Hammerhead ribozyme (HHR), so we decided to construct our genetic device. But however, during the process we realized that the downstream element in EnvZ, about 30 bp before the stop codon of EnvZ, was missing in BBa_K769000. Member from last year did not realize this because they were using the sequence of cph8 for sequence analysis, and did not search against the whole EnvZ sequence. We realized this mid-September, so we quickly re-constructed our sensory-rhodopin fused with EnvZ by designing the following primers:<br />
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<p align=center><imsg src=https://static.igem.org/mediawiki/2013/c/cf/Primers2.jpg></p><br />
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The obtained PCR product was ligated into pSB1A3. Pconst(J23100) and RBS(B0034) were added infront of our new sensory rhodopin fused with EnvZ, and double terminator was added (B0015). This plasmid, together with pSB1C3-PompC-RBS-GFP-doubleTerm. was used to transform <i>E. coli</i> (<i>ΔEnvZ</I>). The colony was confirmed by colony PCR, and was pre-cultured for 12 hours. They were then inoculated into 2 mL LB medium. Half of the samples prepared were covered by aluminium foil and the other half were subjected to light for 12 hours.<br />
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<BR>After 12 hours of culturing under light or wrapped in aluminium foil, the OD at 595 nm and GFP fluorescence was measured. The result is shown below:<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg width=600><br />
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<p align=center>Fig.4 Graph showing the result of light illuminated or non-illuminated culture. <i>E. coli</i> (<i>ΔEnvZ</I>) harboring two plasmids: pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm.</p><br />
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<BR>As you can see, under the light sensoryrhodopsin-EnvZ enhanced GFP expression and under the dark, it repressed GFP expression. This is a light sensor that can enhance gene expression under light suppress it under dark. However, in practice we do not want high background, meaning that we do not want gene expression leakage in the dark. <br />
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<BR>The graph below shows GFP expression comparison with <i>E. coli</i> (<i>ΔEnvZ</I>) harboring ONLY pSB1C3-PompC-RBS-GFP-doubleTerm, without sensory rhodopsin.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2013/1/15/Graph1.jpg width=600></p><br />
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<p align=center>Fig.5 Graph showing the result of <i>E. coli</i> (<i>ΔEnvZ</I>) harboring pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm under light or dark, and <i>E. coli</i> (<i>ΔEnvZ</I>) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm. </p><br />
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<i>E. coli</i> (<i>ΔEnvZ</I>) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm is showing a very high background, which is an indication that probably EnvZ histidine kinase is activated by OmpR from another factor inside the cell. If we want to try and strictly control the gene expression, we might have to use <i>E. coli</i> (<i>ΔEnvZ</I>) with OmpR delted mutation.<br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
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2013-09-28T00:51:46Z
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg width=500></p><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=600></p><br />
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<p align=center><br />
Fig.5 Comparison of normalized GFPuv fluorescence under white light condition and dark condition.<br />
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Sensory rhodopsin : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1C3-<i>Pconst.-SRII-HtrII-EnvZ-PompC-GFP</i><br />
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Negative control : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1A3-<i>PompC-GFP</i><br />
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Positive control : <i>E.coli</i> BL21(DE3)/pSB1A3-<i>PompC-GFP</i> </p><br />
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<BR>As you can see from the result, we obtained a sensory rhodopsin fused by EnvZ that enhanced the down stream expression in the dark and repressed expression under light. For this year's project, we wanted to combine this light sensor with our Hammerhead ribozyme (HHR), so we decided to construct our genetic device. But however, during the process we realized that the downstream element in EnvZ, about 30 bp before the stop codon of EnvZ, was missing in BBa_K769000. Member from last year did not realize this because they were using the sequence of cph8 for sequence analysis, and did not search against the whole EnvZ sequence. We realized this mid-September, so we quickly re-constructed our sensory-rhodopin fused with EnvZ by designing the following primers:<br />
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<p align=center><imsg src=https://static.igem.org/mediawiki/2013/c/cf/Primers2.jpg></p><br />
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The obtained PCR product was ligated into pSB1A3. Pconst(J23100) and RBS(B0034) were added infront of our new sensory rhodopin fused with EnvZ, and double terminator was added (B0015). This plasmid, together with pSB1C3-PompC-RBS-GFP-doubleTerm. was used to transform <i>E. coli</i> (<i>ΔEnvZ</I>). The colony was confirmed by colony PCR, and was pre-cultured for 12 hours. They were then inoculated into 2 mL LB medium. Half of the samples prepared were covered by aluminium foil and the other half were subjected to light for 12 hours.<br />
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<BR>After 12 hours of culturing under light or wrapped in aluminium foil, the OD at 595 nm and GFP fluorescence was measured. The result is shown below:<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg width=600><br />
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<p align=center>Fig.4 Graph showing the result of light illuminated or non-illuminated culture. <i>E. coli</i> (<i>ΔEnvZ</I>) harboring two plasmids: pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm.</p><br />
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<BR>As you can see, under the light sensoryrhodopsin-EnvZ enhanced GFP expression and under the dark, it repressed GFP expression. This is a light sensor that can enhance gene expression under light suppress it under dark. However, in practice we do not want high background, meaning that we do not want gene expression leakage in the dark. <br />
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<BR>The graph below shows GFP expression comparison with <i>E. coli</i> (<i>ΔEnvZ</I>) harboring ONLY pSB1C3-PompC-RBS-GFP-doubleTerm, without sensory rhodopsin.<br />
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<img src=https://static.igem.org/mediawiki/2013/1/15/Graph1.jpg width=600><br />
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<p align=center>Fig.5 Graph showing the result of <i>E. coli</i> (<i>ΔEnvZ</I>) harboring pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm under light or dark, and <i>E. coli</i> (<i>ΔEnvZ</I>) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm. </p><br />
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<i>E. coli</i> (<i>ΔEnvZ</I>) harboring only pSB1C3-PompC-RBS-GFP-doubleTerm is showing a very high background, which is an indication that probably EnvZ histidine kinase is activated by OmpR from another factor inside the cell. If we want to try and strictly control the gene expression, we might have to use <i>E. coli</i> (<i>ΔEnvZ</I>) with OmpR delted mutation.<br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:48:45Z
<p>Miyake: </p>
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg width=500></p><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=600></p><br />
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Fig.5 Comparison of normalized GFPuv fluorescence under white light condition and dark condition.<br />
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Sensory rhodopsin : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1C3-<i>Pconst.-SRII-HtrII-EnvZ-PompC-GFP</i><br />
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Negative control : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1A3-<i>PompC-GFP</i><br />
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Positive control : <i>E.coli</i> BL21(DE3)/pSB1A3-<i>PompC-GFP</i> </p><br />
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<BR>As you can see from the result, we obtained a sensory rhodopsin fused by EnvZ that enhanced the down stream expression in the dark and repressed expression under light. For this year's project, we wanted to combine this light sensor with our Hammerhead ribozyme (HHR), so we decided to construct our genetic device. But however, during the process we realized that the downstream element in EnvZ, about 30 bp before the stop codon of EnvZ, was missing in BBa_K769000. Member from last year did not realize this because they were using the sequence of cph8 for sequence analysis, and did not search against the whole EnvZ sequence. We realized this mid-September, so we quickly re-constructed our sensory-rhodopin fused with EnvZ by designing the following primers:<br />
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<p align=center><imsg src=https://static.igem.org/mediawiki/2013/c/cf/Primers2.jpg></p><br />
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The obtained PCR product was ligated into pSB1A3. Pconst(J23100) and RBS(B0034) were added infront of our new sensory rhodopin fused with EnvZ, and double terminator was added (B0015). This plasmid, together with pSB1C3-PompC-RBS-GFP-doubleTerm. was used to transform <i>E. coli</i> (<i>ΔEnvZ</I>). The colony was confirmed by colony PCR, and was pre-cultured for 12 hours. They were then inoculated into 2 mL LB medium. Half of the samples prepared were covered by aluminium foil and the other half were subjected to light for 12 hours.<br />
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<BR>After 12 hours of culturing under light or wrapped in aluminium foil, the OD at 595 nm and GFP fluorescence was measured. The result is shown below:<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg width=600><br />
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<p align=center>Fig.4 Graph showing the result of light illuminated or non-illuminated culture. <i>E. coli</i> (<i>ΔEnvZ</I>) harboring two plasmids: pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm.<br />
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<BR>As you can see, under the light sensoryrhodopsin-EnvZ enhanced GFP expression and under the dark, it repressed GFP expression. This is a light sensor that can enhance gene expression under light suppress it under dark. However, in practice we do not want high background, meaning that we do not want gene expression leakage in the dark. <br />
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<BR>The graph below shows GFP expression comparison with <i>E. coli</i> (<i>ΔEnvZ</I>) harboring ONLY pSB1C3-PompC-RBS-GFP-doubleTerm, without sensory rhodopsin.<br />
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<img src=https://static.igem.org/mediawiki/2013/1/15/Graph1.jpg width=600><br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:46:00Z
<p>Miyake: </p>
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
<BR><br />
<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
<BR><br />
<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg width=500></p><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=600></p><br />
<BR><br />
<p align=center><br />
Fig.5 Comparison of normalized GFPuv fluorescence under white light condition and dark condition.<br />
<BR><br />
Sensory rhodopsin : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1C3-<i>Pconst.-SRII-HtrII-EnvZ-PompC-GFP</i><br />
<BR><br />
Negative control : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1A3-<i>PompC-GFP</i><br />
<BR><br />
Positive control : <i>E.coli</i> BL21(DE3)/pSB1A3-<i>PompC-GFP</i> </p><br />
<BR><br />
<BR><br />
<BR>As you can see from the result, we obtained a sensory rhodopsin fused by EnvZ that enhanced the down stream expression in the dark and repressed expression under light. For this year's project, we wanted to combine this light sensor with our Hammerhead ribozyme (HHR), so we decided to construct our genetic device. But however, during the process we realized that the downstream element in EnvZ, about 30 bp before the stop codon of EnvZ, was missing in BBa_K769000. Member from last year did not realize this because they were using the sequence of cph8 for sequence analysis, and did not search against the whole EnvZ sequence. We realized this mid-September, so we quickly re-constructed our sensory-rhodopin fused with EnvZ by designing the following primers:<br />
<BR><br />
<p align=center><imsg src=https://static.igem.org/mediawiki/2013/c/cf/Primers2.jpg></p><br />
<BR><br />
<BR><br />
The obtained PCR product was ligated into pSB1A3. Pconst(J23100) and RBS(B0034) were added infront of our new sensory rhodopin fused with EnvZ, and double terminator was added (B0015). This plasmid, together with pSB1C3-PompC-RBS-GFP-doubleTerm. was used to transform <i>E. coli</i> (<i>ΔEnvZ</I>). The colony was confirmed by colony PCR, and was pre-cultured for 12 hours. They were then inoculated into 2 mL LB medium. Half of the samples prepared were covered by aluminium foil and the other half were subjected to light for 12 hours.<br />
<BR><br />
<BR>After 12 hours of culturing under light or wrapped in aluminium foil, the OD at 595 nm and GFP fluorescence was measured. The result is shown below:<br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg width=600><br />
<BR><br />
<p align=center>Fig.4 Graph showing the result of light illuminated or non-illuminated culture. <i>E. coli</i> (<i>ΔEnvZ</I>) harboring two plasmids: pSB1A3-Pconst(H)-RBS-rhodopsinEnvZ-doubleTerm and pSB1C3-PompC-RBS-GFP-doubleTerm.<br />
<BR><br />
<BR.<br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/1/15/Graph1.jpg><br />
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<BR><br />
<BR><br />
<BR><br />
Reference<br />
<BR><br />
<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:44:00Z
<p>Miyake: </p>
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
<BR><br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
<BR><br />
<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<font size=4><br />
<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<BR><br />
<BR><br />
<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
<BR><br />
<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg width=500></p><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=600></p><br />
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<p align=center><br />
Fig.5 Comparison of normalized GFPuv fluorescence under white light condition and dark condition.<br />
<BR><br />
Sensory rhodopsin : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1C3-<i>Pconst.-SRII-HtrII-EnvZ-PompC-GFP</i><br />
<BR><br />
Negative control : <i>E.coli</i> MG1655(Δ<i>EnvZ</i>)/pSB1A3-<i>PompC-GFP</i><br />
<BR><br />
Positive control : <i>E.coli</i> BL21(DE3)/pSB1A3-<i>PompC-GFP</i> </p><br />
<BR><br />
<BR><br />
<BR>As you can see from the result, we obtained a sensory rhodopsin fused by EnvZ that enhanced the down stream expression in the dark and repressed expression under light. For this year's project, we wanted to combine this light sensor with our Hammerhead ribozyme (HHR), so we decided to construct our genetic device. But however, during the process we realized that the downstream element in EnvZ, about 30 bp before the stop codon of EnvZ, was missing in BBa_K769000. Member from last year did not realize this because they were using the sequence of cph8 for sequence analysis, and did not search against the whole EnvZ sequence. We realized this mid-September, so we quickly re-constructed our sensory-rhodopin fused with EnvZ by designing the following primers:<br />
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<p align=center><imsg src=https://static.igem.org/mediawiki/2013/c/cf/Primers2.jpg></p><br />
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<BR><br />
The obtained PCR product was ligated into pSB1A3. Pconst(J23100) and RBS(B0034) were added infront of our new sensory rhodopin fused with EnvZ, and double terminator was added (B0015). This plasmid, together with pSB1C3-PompC-RBS-GFP-doubleTerm. was used to transform <i>E. coli</i> (<i>ΔEnvZ</I>). The colony was confirmed by colony PCR, and was pre-cultured for 12 hours. They were then inoculated into 2 mL LB medium. Half of the samples prepared were covered by aluminium foil and the other half were subjected to light for 12 hours.<br />
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<BR>After 12 hours of culturing under light or wrapped in aluminium foil, the OD at 595 nm and GFP fluorescence was measured. The result is shown below:<br />
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<img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg><br />
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<img src=https://static.igem.org/mediawiki/2013/1/15/Graph1.jpg><br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/File:Primers2.jpg
File:Primers2.jpg
2013-09-28T00:38:39Z
<p>Miyake: </p>
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:28:52Z
<p>Miyake: </p>
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<ul id="contents"><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<font size=4><br />
<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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<BR><br />
<BR><br />
Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
<BR><br />
<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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<img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg><br />
<BR><br />
<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=600></p><br />
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<p align=center>Graph showing GFP expression <br />
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<img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg><br />
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<img src=https://static.igem.org/mediawiki/2013/1/15/Graph1.jpg><br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:27:30Z
<p>Miyake: </p>
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>We experssed the following construct inside <i>E. coli</i> (<i>ΔEnvZ</I>) and measured GFP fluorescence.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/d/d6/Rhodopsin3.jpg/800px-Rhodopsin3.jpg width=400></p><br />
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<BR>This is the result.<br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png width=800></p><br />
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<p align=center>Graph showing GFP expression <br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:24:18Z
<p>Miyake: </p>
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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<BR>This is our result from last year. <br />
<p align=center><img src=https://static.igem.org/mediawiki/2012/thumb/1/16/Rhodopsinfig5.png/711px-Rhodopsinfig5.png></p><br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:22:35Z
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg><br />
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<img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg><br />
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<BR><p align=center>Fig.2 Construct from Tokyo-NoKogen 2012. EnvZ hisidine kinase domain was obtained from cph8, cut at <i>Nde</i>I and Spe<i>I</i> and fused together.<br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:17:12Z
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<td class="home"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen"><span class="home"></span></a></td><br />
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<td class="project1"><div class="project1"></div></td><br />
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<td class="team"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Team"><span class="team"></span></a></td><br />
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<td class="biobrick"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Parts"><span class="biobrick"></span></a></td><br />
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<td class="notebook"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Notebook"><span class="notebook"></span></a></td><br />
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<td class="humanpractice"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Humanpractice"><span class="humanpractice"></span></a></td> <br />
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<td class="achievement"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Achievement"><span class="achievement"></span></a></td><br />
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<div id="index"><br />
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<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) are known to show phototaxis by the illumination of light by using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. This two-component system regulates cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
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<p align=center><img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg></p><br />
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<p align=center>Fig.1 Sensory rhodopsin II (SRII) (orange) from N. pharaonis, fused to cognate transducer protein HtrII (green) by a 9 amino acid linker, fused to E. coli chemotaxis receptor Tsr (blue) in the cytoplasmic region. (Ref.4) </p><br />
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It has been previously reported that chimeric rhodopsin can be constructed inside <i>E. coli</I>. Kwang-Hwan <i>et al.</i> constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from <i>N. pharaonis</i> with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a that <i>E. coli</I> succesfully responded to light (Ref.3). It was also previously reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. <br />
So last year, Tokyo-NoKoGen 2012 have deduced that sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg><br />
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<img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg><br />
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<img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg><br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:11:47Z
<p>Miyake: </p>
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<div id="index"><br />
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<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
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<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<BR>Last year's team member ofTokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>. <br />
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Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis (N. pharaonis) show phototaxis by responding to changes in light color and intensity using receptors called sensory rhodopsin I and II (SRI and SRII). The SR proteins are seven-transmembrane retinylidene photoreceptors, which transmits blue light signal (λmax 487 nm) to their corresponding transducers HtrI and HtrII respectively. signals to Htr proteins via helix-helix interaction. Htr protein consists of two transmembrane helices and a cytoplasmic methyl-accepting and His-Kinase domain, and belongs to histidine kinase / phosphoreregulator two-component system for regulating cells’ flagellar motors for phototaxis (Ref. 1, 2). <br />
<BR><br />
It has been previously investigated to see how chimeric rhodopsin responds to light signals inside Escherichia coli (E. coli). Kwang-Hwan et al. constructed chimeric transducer proteins, by exchanging the Histidine-kinase domain of the bacteriorhodopsin from N. pharaonis with homologous chemotaxis transducers Tsr and Tar of E. coli and Salmonella enterica, (chemotaxis transducers of serine and aspartate respectively).They observed a phototaxis response of E. coli showed that it responds to light (Ref.3). It was also reported, that the sensory domain of Tar protein fused with histidine kinase domain of EnvZ protein of E. coli, functioned as a sensor protein to transmit signal received by the Tar sensory domain to the EnvZ two-component system (Ref.5). The histidine kinase domain of Tar protein and EnvZ protein are homologous. This year, we have deduced from the reported results, that because a fusion protein Tar-EnvZ functions inside E. coli, sensory domain of sensory rhodopsin fused with EnvZ protein should also function to transmit signal downstream of the two-component system. <br />
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<img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg><br />
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<img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg><br />
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Reference<br />
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<BR>[1] Xue-Nong Zhang et al. (1999) The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices, Proc. Natl. Acad. Sci. USA <br />
<BR>[2] Wouter D. Hoff et al. (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsin, Anmu. Rev. Biophys. Biomol. Struct. <br />
<BR>[3] Kwang-Hwan Jung et al. (2001) An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli, Journal of bacteriology <br />
<BR>[4] Vishwa D. et al. (2003) Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phototransfer in vitro, Biochemistry <br />
<BR>[5] Yoshida T et al., (2007) The design and development of Tar-EnvZ chimeric receptors, Methods Enzymol.<br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:09:32Z
<p>Miyake: </p>
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<br />
</div><br />
<br />
<div id="index"><br />
<br />
<br />
<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
<br />
</ul><br />
<br />
</div><br />
<br />
<div id="main"><br />
<BR><br />
<BR><br />
<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
<BR><br />
<br />
<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
<BR><br />
<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
<br />
<BR><br />
<br />
<BR>Last year's team member of out team, Tokyo-NoKoGen2012 have been working on developing a light sensor by combining the sensory domain of rhodospin derived from <i>N. pharaonis</I>, and the histidine kinase domain of EnvZ from <I>Escherichia coli</I>.<br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg><br />
<BR><br />
<BR><br />
<br />
<img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg><br />
<BR><br />
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<br />
<img src=https://static.igem.org/mediawiki/2013/8/8d/Graphb.jpg><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:07:09Z
<p>Miyake: </p>
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<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
<br />
</ul><br />
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<br />
<div id="main"><br />
<BR><br />
<BR><br />
<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
<BR><br />
<br />
<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
<BR><br />
<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
<br />
<BR><br />
<br />
<BR><br />
<BR><br />
<imt src=https://static.igem.org/mediawiki/2012/b/b9/Rhodopsin1.jpg><br />
<BR><br />
<BR><br />
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<img src=https://static.igem.org/mediawiki/2012/thumb/c/cb/Rhodopsin2.jpg/800px-Rhodopsin2.jpg><br />
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<li class="LN"><a target="_blank" href="http://lne.st/"><img class="LN" src="https://static.igem.org/mediawiki/2013/2/2a/リバネス.png"></a></li><br />
<li class="IR"><a target="_blank" href="http://www.ikedarika.co.jp/english/"><img class="IR" src="https://static.igem.org/mediawiki/2013/b/b8/池田理化.gif"></a></li><br />
<li class="Promega"><a target="_blank" href="http://www.promega.com/"><img class="Progma" src="https://static.igem.org/mediawiki/2013/c/c4/Promega.jpg"></li><br />
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<br />
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<li class="IK"><a target="_blank" href="http://www.tuat.ac.jp/~kakusan/index.html"><img src="https://static.igem.org/mediawiki/2013/8/87/池袋研究室.png"></a></li><br />
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<br />
<br />
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<br />
</body><br />
<br />
</html></div>
Miyake
http://2013.igem.org/File:Graph1.jpg
File:Graph1.jpg
2013-09-28T00:05:57Z
<p>Miyake: </p>
<hr />
<div></div>
Miyake
http://2013.igem.org/File:Graphb.jpg
File:Graphb.jpg
2013-09-28T00:05:09Z
<p>Miyake: </p>
<hr />
<div></div>
Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:04:23Z
<p>Miyake: </p>
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<br />
<div id="mymenubar"><br />
<br />
<table><br />
<tbody><br />
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<td class="home"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen"><span class="home"></span></a></td><br />
<br />
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<div id="index"><br />
<br />
<br />
<h1 id="index_title"></h1><br />
<ul id="contents"><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
<br />
</ul><br />
<br />
</div><br />
<br />
<div id="main"><br />
<BR><br />
<BR><br />
<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
<br />
<p align=center><font size=5>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
<BR><br />
<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
<br />
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<li class="UI"><a target="_blank" href="http://www.ultizyme.jp/"><img class="UI" src="https://static.igem.org/mediawiki/2013/4/47/アルティザイム・インターナショナル.jpg" ></a></li><br />
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<li class="LN"><a target="_blank" href="http://lne.st/"><img class="LN" src="https://static.igem.org/mediawiki/2013/2/2a/リバネス.png"></a></li><br />
<li class="IR"><a target="_blank" href="http://www.ikedarika.co.jp/english/"><img class="IR" src="https://static.igem.org/mediawiki/2013/b/b8/池田理化.gif"></a></li><br />
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<li class="NK"><a target="_blank" href="http://www.tuat.ac.jp/en/index.html"><img class ="NK"src="https://static.igem.org/mediawiki/2013/7/76/東京農工大学.png"></a></li><br />
<li class="ST"><a target="_blank" href="http://www.tuat.ac.jp/~tanpaku/"><img src="https://static.igem.org/mediawiki/2013/5/5a/早出・津川研究室.png" ></a></li><br />
<li class="IK"><a target="_blank" href="http://www.tuat.ac.jp/~kakusan/index.html"><img src="https://static.igem.org/mediawiki/2013/8/87/池袋研究室.png"></a></li><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:03:28Z
<p>Miyake: </p>
<hr />
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<h1 id="index_title"></h1><br />
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
<br />
</ul><br />
<br />
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<br />
<div id="main"><br />
<BR><br />
<BR><br />
<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
<br />
<BR>Sensory Rhodopsin fused with EnvZ histidine kinase (BBa_K769000) <br />
<BR><br />
<p align=center><font size=3>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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<li class="ST"><a target="_blank" href="http://www.tuat.ac.jp/~tanpaku/"><img src="https://static.igem.org/mediawiki/2013/5/5a/早出・津川研究室.png" ></a></li><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-28T00:01:09Z
<p>Miyake: </p>
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
<br />
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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<BR><font size=4>From Tokyo-NoKoGen 2012 https://2012.igem.org/Team:Tokyo-NoKoGen</font><br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin
Team:Tokyo-NoKoGen/rhodopsin
2013-09-27T23:58:07Z
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<p align=center><font size=6>Improving a BioBrick part</p></font> <br />
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Miyake
http://2013.igem.org/Team:Tokyo-NoKoGen/Project
Team:Tokyo-NoKoGen/Project
2013-09-27T23:57:00Z
<p>Miyake: </p>
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<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator"><li><a href="#diary"><strong>RNA oscillator</a></strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold"><li><strong>RNA scaffold</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/light"><li><strong>Light sensor</strong></li></a><br />
<a href="https://2013.igem.org/Team:Tokyo-NoKoGen/modeling"><li><strong>Modeling</strong></li></a><br />
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<p align=center><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/overview>Overview</a></p> <br />
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<p align=center><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/oscillator>RNA oscillator</a></p> <br />
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<p align=center><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/scaffold>RNA Scaffold</a></p> <br />
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<p align=center><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/light>Light sensor</a></p> <br />
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<p align=center><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/modeling>Modeling</a></p><br />
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<p align=center><a href=https://2013.igem.org/Team:Tokyo-NoKoGen/rhodopsin>Improving a BioBrick part - Rhodopsin</a></p><br />
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<li class="LN"><a target="_blank" href="http://lne.st/"><img class="LN" src="https://static.igem.org/mediawiki/2013/2/2a/リバネス.png"></a></li><br />
<li class="IR"><a target="_blank" href="http://www.ikedarika.co.jp/english/"><img class="IR" src="https://static.igem.org/mediawiki/2013/b/b8/池田理化.gif"></a></li><br />
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<li class="NK"><a target="_blank" href="http://www.tuat.ac.jp/en/index.html"><img class ="NK"src="https://static.igem.org/mediawiki/2013/7/76/東京農工大学.png"></a></li><br />
<li class="ST"><a target="_blank" href="http://www.tuat.ac.jp/~tanpaku/"><img src="https://static.igem.org/mediawiki/2013/5/5a/早出・津川研究室.png" ></a></li><br />
<li class="IK"><a target="_blank" href="http://www.tuat.ac.jp/~kakusan/index.html"><img src="https://static.igem.org/mediawiki/2013/8/87/池袋研究室.png"></a></li><br />
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Miyake