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http://2013.igem.org/Team:Tokyo-NoKoGen/oscillator
Team:Tokyo-NoKoGen/oscillator
2013-10-16T09:20:54Z
<p>Jinhee: </p>
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<title>Team:Tokyo-NoKoGen - 2013.igem.org</title><br />
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<link rel="stylesheet" type="text/css" href="Tokyo-NoKoGen.css"><br />
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</head><br />
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<body><br />
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<div id="wrapper"><br />
<br />
<div id="title_logo"> <br />
<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 />
<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 />
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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 />
</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></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="#TR-HHR">TR-HHR</a></li><br />
<li><a href="#TR-HHR-taRNA">TR-HHR-taRNA</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 />
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</div><br />
<div id="main"><br />
<br />
<font size=5><br />
<BR><br />
<p align=center><font size=7><strong>RNA oscillator</strong></font><hr></p><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6><strong>Introduction </span></strong></font><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6 ><strong>Protein oscillator</span></strong></font><br />
<BR><br />
<p style="line-height:110%"><br />
<BR><br />
The oscillating network was built by using three transcriptional repressor systems in which the first repressor protein suppresses the expression of second repressor gene, whose protein suppresses the transcription of the third gene, and the protein the third gene code suppresses the first gene expression.<br />
</p><br />
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<BR><br />
<BR><br />
<br />
<font size=6><strong>Hummer headed ribozyme</span></strong></font><br />
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<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
Hummerheaded ribozymes (HHRs) are found in many species and known to have a capability to self-cleave. Recently, engineered HHRs responding to trans-acting RNAs (taRNAs) in <I>E.coli</I> were reported. In absence of taRNA, the HHRs can self-cleave, but in presence of taRNA, their capability of self-cleavage is inhibited due to hybridize with taRNA and change their structures.<br />
</p><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/3a/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A318.PNG><BR><br />
<br />
<BR><BR><BR><br />
<br />
<p style="line-height:110%"><br />
We developed the new oscillator system that is composed only RNAs not using repressor gene and protein. We created three taRNA connected hummerheaded ribozymes (HHR-taRNA) that sense to another taRNA. The same way as the protein oscillator, three HHR-taRNAs inhibits other one’s self-cleavage such as three-way standoff. When the first HHR-taRNA is self-cleaved, connected the first taRNA that binds to the second HHR-taRNA is free and suppresses the second HHR-taRNA self-cleavage. When second HHR-taRNA is self-cleaved, the second taRNA that binds to the third HHR-taRNA is free and suppresses the third HHR-taRNA. Finally, when third HHR-taRNA is self-cleaved, the third taRNA that binds to the first HHR-taRNA is free and suppresses the first HHR-taRNA, completing cycle.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
<font size=6 id="Objective"><strong>Objective</span></strong></font><br />
<BR><br />
<BR><br />
Construction of Three of orthogonal taRNA generator which repressed by another taRNA.<br />
<br />
<BR><br />
<br />
<p style="line-height:110%"><br />
taRNA responsive HHR (TR-HHR) was fused with taRNA. By using orthogonal riboregulator sequences, we designed three of taRNA generators.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
<font size=6 id="Method"><strong>Method</span></strong></font><br />
<br />
<BR><BR><br />
<br />
<font size=5 id="Design"><strong>-Design</span></strong></font> <br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
TR-HHR was designed by Benedikt Klauser and Jo¨ rg S. Hartig. Their TR-HHR were inspired by the riboregulatory system previously reported by Collins and coworkers. They made use of sequence domains of the riboregulator RR12. And, orthogonal variants of riboregulator RR12 (RR12y, RR42) were reported by Collins and coworkers. So, by using the sequences of these orthogonal riboregulators, we designed three orthogonal TR-HHRs.<br />
</p><br />
<br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a1/Rrvariants.png" width="400px" / ><br />
<img src="https://static.igem.org/mediawiki/2013/c/ce/Cccc.png" width="200px" / ><br />
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<BR><BR><br />
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<BR><BR><BR><br />
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<font size=5 id="Parts construction"><strong>-Parts construction</span></strong></font> <br />
<BR><br />
<p style="line-height:110%"><br />
1. Construction of TR-HHR<BR><br />
2. Construction of TR-HHR variants<BR><br />
3. Construction of taRNA variants<BR><br />
4. Construction of TR-HHR-taRNA<BR><br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
1. Construction of TR-HHR [BBa_K1053000]<BR><br />
<br />
<p style="line-height:110%"> <br />
We used the TR-HHR developed by Klauser <I>et al.</I>[1] Two overlap primers designed, and PCR was performed to connect them. The PCR product was ligated into pSB1C3 vector. <br />
</p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/e/e3/Wiki1.png" width="400px" / ><br />
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<BR><BR><br />
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2.Construction of TR-HHR variants<br />
<br />
<BR><BR><BR><br />
<br />
<p style="line-height:110%"><br />
By inverse PCR using pSB1C3-TR-HHR as a template, we could obtain three TR-HHRs, TR(12)-HHR, TR(12y)-HHR, and TR(42)-HHR.<br />
</p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/9/98/Wiki6.png" width="400px" / ><br />
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<BR><br />
<BR><br />
<BR><br />
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3.Construction of taRNA variants<br />
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<BR><br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
By inverse PCR using pSB1A3-taR12-DT as a template, we could obtain three taRNAs, taR12, taR12y, taR42.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a7/Wiki2.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<br />
4.Construction of TR-HHR-taRNA<br />
<BR><BR><br />
[BBa_K1053006][BBa_K1053015][BBa_K1053016]<br />
<BR><br />
<BR><br />
<br />
TR-HHR-taRNAs were constructed by overlap PCR.<br />
<br />
<BR><br />
<BR><br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/9/91/Wiki3.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6 id="Evaluation"><strong>Evaluation</strong></font><br />
<BR><br />
<BR><br />
I. Evaluation of TR-HHR<br />
<BR><BR><br />
II. Evaluation of TR-HHR-taRNA<br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="TR-HHR"><strong>I. Evaluation of TR-HHR</strong></font><br />
<br />
<BR><br />
<br />
<p style="line-height:110%"><br />
In order to evaluate the regulation of HHR’s self-cleavage activity by taRNA, GFP gene was connected with the HHR or inactive HHR (*HHR), which has a mutation lose the activity.[BBa_K1053004][BBa_K1053005]<BR> These fragments were subcloned into conventional biobrick standard vector pSB3C5. <br />
</p><br />
<br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/4/45/Wiki4.png" width="400px" / ><br />
<BR><br />
<br />
1) <I>E. coli</I> TOP10 strain was co-transformed by two plasmids listed bellow, <br />
<BR><br />
<BR><br />
A)<br />
<BR><br />
pSB1A3-taR12-DT<br />
<BR><br />
pSB3C5-TR(12)-HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
B)<br />
<BR><br />
pSB1A3-empty<br />
<BR><br />
pSB3C5- TR(12)-HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
C) <br />
<BR><br />
pSB1A3-taR12-DT<br />
<BR><br />
pSB3C5-TR(12)- * HHR-GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
D)<br />
<BR><br />
pSB1A3 empty<br />
<BR><br />
pSB3C5- TR(12)- * HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
E) <br />
<BR>pSB1A3-empty<br />
<BR><br />
pSB3C5-empty<br />
<br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
2) Bacterial cultures were incubated in LB medium at 140 rpm and 37℃ for 12 hour. <BR><br />
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.<BR><br />
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.<BR><br />
5) OD595 and GFP fluorescence intensity were measured.<BR><br />
All experiments were performed using three cultures per each sample.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
Result and Discussion<br />
<br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/f/fb/%E3%82%B9%E3%83%A9%E3%82%A4%E3%83%897.jpg width="400px"><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
In the presence of taR12, GFP fluorescence value divided by OD value is higher than one in absence of taR12. This result was not expected. HHR has the ability to self-cleave, but its ability is suppressed when taRNA hybridizes to HHR. Therefore, in the presence of taRNA, self-cleavage of HHR should not happen. So then, why we get such result? One factor can be due to the exposure of RBS sequence caused by the structural change of HHR when it hybridizes to taRNA<br />
</p><br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="TR-HHR-taRNA">II.Evaluation of TR-HHR-taRNA</font><br />
<br />
<BR><BR><br />
<br />
We examined whether TR-HHR-taRNA’s output can be repressed by corresponding taRNA. <br />
<BR><br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/5/52/Wiki5.png" width="400px" / ><br />
<BR><br />
<br />
<br />
1) The plasmids as shown below was used to transform <I>E. coli </I>Top10.<BR><br />
<BR><br />
A) <br />
<BR><br />
pSB1A3-P<sub>bad</sub> -TR(42)HHR-taR12-DT<br />
<BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
B) <br />
<BR><br />
pSB1A3-P<sub>const(H)</sub>-taR42-P<sub>bad</sub>-TR(42)HHR-taR12-DT<BR><br />
pSB3C5-crR12-GFP-DT<br />
<br />
<BR><BR><BR><br />
<br />
C)<br />
<BR><br />
pSB1A3-P<sub>bad</sub>-taR12-DT<BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
D) <BR><br />
pSB1A3-empty <BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
E) <BR><br />
pSB1A3-empty<BR><br />
pSB3C5-empty<BR><br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
2) The transformants were pre-cultured in LB medium at 37 degrees celcius for 8 hours. <BR><br />
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.<BR><br />
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.<BR><br />
5) OD595 and GFP fluorescence intensity were measured.<BR><br />
</p><br />
<br />
<BR><BR><br />
<br />
<font size=5 ><strong>Result and Discussion</strong></font><br />
<br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/c/c3/%E5%AE%9F%E9%A8%93%EF%BC%92%E7%B5%90%E6%9E%9C%EF%BC%91.jpg width="400px"><br />
<BR><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/6/69/%E5%AE%9F%E9%A8%93%EF%BC%92%E7%B5%90%E6%9E%9C%EF%BC%92.jpg width="400px"><br />
<BR><br />
<p style="line-height:110%"><br />
In order to evaluate a function of TR(42)-HHR-taR12, we used crR12-GFP as a reporter.<br />
taRNA is complementary to the sequence of crRNA, and by the binding of taRNA to crRNA, RBS is exposed and gene expression is activated. In this result, the GFP fluorescence value divided by OD value of (2) was lower than that of (3). It shows that taR42 suppressed self-cleavage of TR(42)-HHR and caused less GFP expression. Therefore, we prove that the self-cleavage activity of TR(42)-HHR and amount of taR12 can be controlled by taR42.<br />
</p><br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="Future work" ><strong>・Future work</strong></font><br />
<br />
<BR><br />
We must confirm whether taR12 and TR(12)-HHR set is available for RNA oscillator.<BR><br />
The second experiment result showed high background, then we should try to use lower promoter to transcribe crR12-GFP.<BR><br />
<BR><br />
<br />
<font size=6 ><strong>Reference</strong></font><br />
<br />
<BR><BR><br />
<BR><br />
<p style="line-height:110%"><br />
[1]<br />
An engineered small RNA-mediated genetic switch<br />
based on a ribozyme expression platform<br />
Benedikt Klauser and Jo¨ rg S. Hartig <br />
Nucleic Acids Research, 2013<br />
<BR><br />
[2]<br />
Callura,J.M., Cantor,C.R. and Collins,J.J. (2012) Genetic<br />
switchboard for synthetic biology applications. Proc. Natl Acad.<br />
Sci. USA, 109, 5850–5855<br />
</p><br />
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<BR><br />
<br />
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Jinhee
http://2013.igem.org/Team:Tokyo-NoKoGen/oscillator
Team:Tokyo-NoKoGen/oscillator
2013-09-28T04:04:54Z
<p>Jinhee: </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="#TR-HHR">TR-HHR</a></li><br />
<li><a href="#TR-HHR-taRNA">TR-HHR-taRNA</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 />
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<div id="main"><br />
<br />
<font size=5><br />
<BR><br />
<p align=center><font size=7><strong>RNA Oscillator</strong></font><hr></p><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6><strong>Introduction </span></strong></font><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6 ><strong>Protein oscillator</span></strong></font><br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
<BR><br />
The oscillating network was built by using three transcriptional repressor systems in which the first repressor protein suppresses the expression of second repressor gene, whose protein suppresses the transcription of the third gene, and the protein the third gene code suppresses the first gene expression.<br />
</p><br />
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<BR><br />
<BR><br />
<br />
<font size=6><strong>Hummer headed ribozyme</span></strong></font><br />
<br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
Hummerheaded ribozymes (HHRs) are found in many species and known to have a capability to self-cleave. Recently, engineered HHRs responding to trans-acting RNAs (taRNAs) in <I>E.coli</I> were reported. In absence of taRNA, the HHRs can self-cleave, but in presence of taRNA, their capability of self-cleavage is inhibited due to hybridize with taRNA and change their structures.<br />
</p><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/3a/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A318.PNG><BR><br />
<br />
<BR><BR><BR><br />
<br />
<p style="line-height:110%"><br />
We developed the new oscillator system that is composed only RNAs not using repressor gene and protein. We created three taRNA connected hummerheaded ribozymes (HHR-taRNA) that sense to another taRNA. The same way as the protein oscillator, three HHR-taRNAs inhibits other one’s self-cleavage such as three-way standoff. When the first HHR-taRNA is self-cleaved, connected the first taRNA that binds to the second HHR-taRNA is free and suppresses the second HHR-taRNA self-cleavage. When second HHR-taRNA is self-cleaved, the second taRNA that binds to the third HHR-taRNA is free and suppresses the third HHR-taRNA. Finally, when third HHR-taRNA is self-cleaved, the third taRNA that binds to the first HHR-taRNA is free and suppresses the first HHR-taRNA, completing cycle.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
<font size=6 id="Objective"><strong>Objective</span></strong></font><br />
<BR><br />
<BR><br />
Construction of Three of orthogonal taRNA generator which repressed by another taRNA.<br />
<br />
<BR><br />
<br />
<p style="line-height:110%"><br />
taRNA responsive HHR (TR-HHR) was fused with taRNA. By using orthogonal riboregulator sequences, we designed three of taRNA generators.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
<font size=6 id="Method"><strong>Method</span></strong></font><br />
<br />
<BR><BR><br />
<br />
<font size=5 id="Design"><strong>-Design</span></strong></font> <br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
TR-HHR was designed by Benedikt Klauser and Jo¨ rg S. Hartig. Their TR-HHR were inspired by the riboregulatory system previously reported by Collins and coworkers. They made use of sequence domains of the riboregulator RR12. And, orthogonal variants of riboregulator RR12 (RR12y, RR42) were reported by Collins and coworkers. So, by using the sequences of these orthogonal riboregulators, we designed three orthogonal TR-HHRs.<br />
</p><br />
<br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a1/Rrvariants.png" width="400px" / ><br />
<img src="https://static.igem.org/mediawiki/2013/c/ce/Cccc.png" width="200px" / ><br />
<br />
<br />
<BR><BR><br />
<br />
<br />
<BR><BR><BR><br />
<br />
<font size=5 id="Parts construction"><strong>-Parts construction</span></strong></font> <br />
<BR><br />
<p style="line-height:110%"><br />
1. Construction of TR-HHR<BR><br />
2. Construction of TR-HHR variants<BR><br />
3. Construction of taRNA variants<BR><br />
4. Construction of TR-HHR-taRNA<BR><br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
1. Construction of TR-HHR<BR><br />
<br />
<p style="line-height:110%"> <br />
We used the TR-HHR developed by Klauser <I>et al.</I>[1] Two overlap primers designed, and PCR was performed to connect them. The PCR product was ligated into pSB1C3 vector.<br />
</p><br />
<br />
<BR><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e3/Wiki1.png" width="400px" / ><br />
<br />
<BR><BR><br />
<br />
2.Construction of TR-HHR variants<br />
<br />
<BR><BR><BR><br />
<br />
<p style="line-height:110%"><br />
By inverse PCR using pSB1C3-TR-HHR as a template, we could obtain three TR-HHRs, TR(12)-HHR, TR(12y)-HHR, and TR(42)-HHR.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/9/98/Wiki6.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<br />
<br />
3.Construction of taRNA variants<br />
<br />
<BR><br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
By inverse PCR using pSB1A3-taR12-DT as a template, we could obtain three taRNAs, taR12, taR12y, taR42.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a7/Wiki2.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<br />
4.Construction of TR-HHR-taRNA<br />
<BR><br />
<BR><br />
<BR><br />
<br />
TR-HHR-taRNAs were constructed by overlap PCR.<br />
<br />
<BR><br />
<BR><br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/9/91/Wiki3.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6 id="Evaluation"><strong>Evaluation</strong></font><br />
<BR><br />
<BR><br />
I. Evaluation of TR-HHR<br />
<BR><BR><br />
II. Evaluation of TR-HHR-taRNA<br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="TR-HHR"><strong>I. Evaluation of TR-HHR</strong></font><br />
<br />
<BR><br />
<br />
<p style="line-height:110%"><br />
In order to evaluate the regulation of HHR’s self-cleavage activity by taRNA, GFP gene was connected with the HHR or inactive HHR (*HHR), which has a mutation lose the activity. These fragments were subcloned into conventional biobrick standard vector pSB3C5. <br />
</p><br />
<br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/4/45/Wiki4.png" width="400px" / ><br />
<BR><br />
<br />
1) <I>E. coli</I> TOP10 strain was co-transformed by two plasmids listed bellow, <br />
<BR><br />
<BR><br />
A)<br />
<BR><br />
pSB1A3-taR12-DT<br />
<BR><br />
pSB3C5-TR(12)-HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
B)<br />
<BR><br />
pSB1A3-empty<br />
<BR><br />
pSB3C5- TR(12)-HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
C) <br />
<BR><br />
pSB1A3-taR12-DT<br />
<BR><br />
pSB3C5-TR(12)- * HHR-GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
D)<br />
<BR><br />
pSB1A3 empty<br />
<BR><br />
pSB3C5- TR(12)- * HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
E) <br />
<BR>pSB1A3-empty<br />
<BR><br />
pSB3C5-empty<br />
<br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
2) Bacterial cultures were incubated in LB medium at 140 rpm and 37℃ for 12 hour. <BR><br />
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.<BR><br />
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.<BR><br />
5) OD595 and GFP fluorescence intensity were measured.<BR><br />
All experiments were performed using three cultures per each sample.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
Result and Discussion<br />
<br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/f/fb/%E3%82%B9%E3%83%A9%E3%82%A4%E3%83%897.jpg width="400px"><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
In the presence of taR12, GFP fluorescence value divided by OD value is higher than one in absence of taR12. This result was not expected. HHR has the ability to self-cleave, but its ability is suppressed when taRNA hybridizes to HHR. Therefore, in the presence of taRNA, self-cleavage of HHR should not happen. So then, why we get such result? One factor can be due to the exposure of RBS sequence caused by the structural change of HHR when it hybridizes to taRNA<br />
</p><br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="TR-HHR-taRNA">II.Evaluation of TR-HHR-taRNA</font><br />
<br />
<BR><BR><br />
<br />
We examined whether TR-HHR-taRNA’s output can be repressed by corresponding taRNA. <br />
<BR><br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/5/52/Wiki5.png" width="400px" / ><br />
<BR><br />
<br />
<br />
1) The plasmids as shown below was used to transform <I>E. coli </I>Top10.<BR><br />
<BR><br />
A) <br />
<BR><br />
pSB1A3-P<sub>bad</sub> -TR(42)HHR-taR12-DT<br />
<BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
B) <br />
<BR><br />
pSB1A3-P<sub>const(H)</sub>-taR42-P<sub>bad</sub>-TR(42)HHR-taR12-DT<BR><br />
pSB3C5-crR12-GFP-DT<br />
<br />
<BR><BR><BR><br />
<br />
C)<br />
<BR><br />
pSB1A3-P<sub>bad</sub>-taR12-DT<BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
D) <BR><br />
pSB1A3-empty <BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
E) <BR><br />
pSB1A3-empty<BR><br />
pSB3C5-empty<BR><br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
2) The transformants were pre-cultured in LB medium at 37 degrees celcius for 8 hours. <BR><br />
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.<BR><br />
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.<BR><br />
5) OD595 and GFP fluorescence intensity were measured.<BR><br />
</p><br />
<br />
<BR><BR><br />
<br />
<font size=5 ><strong>Result and Discussion</strong></font><br />
<br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/c/c3/%E5%AE%9F%E9%A8%93%EF%BC%92%E7%B5%90%E6%9E%9C%EF%BC%91.jpg width="400px"><br />
<BR><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/6/69/%E5%AE%9F%E9%A8%93%EF%BC%92%E7%B5%90%E6%9E%9C%EF%BC%92.jpg width="400px"><br />
<BR><br />
<p style="line-height:110%"><br />
In order to evaluate a function of TR(42)-HHR-taR12, we used crR12-GFP as a reporter.<br />
taRNA is complementary to the sequence of crRNA, and by the binding of taRNA to crRNA, RBS is exposed and gene expression is activated. In this result, the GFP fluorescence value divided by OD value of (2) was lower than that of (3). It shows that taR42 suppressed self-cleavage of TR(42)-HHR and caused less GFP expression. Therefore, we prove that the self-cleavage activity of TR(42)-HHR and amount of taR12 can be controlled by taR42.<br />
</p><br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="Future work" ><strong>・Future work</strong></font><br />
<br />
<BR><BR><BR><br />
<br />
<font size=6 ><strong>Reference</strong></font><br />
<br />
<BR><BR><br />
<BR><br />
<p style="line-height:110%"><br />
[1]<br />
An engineered small RNA-mediated genetic switch<br />
based on a ribozyme expression platform<br />
Benedikt Klauser and Jo¨ rg S. Hartig <br />
Nucleic Acids Research, 2013<br />
<BR><br />
[2]<br />
Callura,J.M., Cantor,C.R. and Collins,J.J. (2012) Genetic<br />
switchboard for synthetic biology applications. Proc. Natl Acad.<br />
Sci. USA, 109, 5850–5855<br />
</p><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Daily_notes
Team:Sumbawagen/Daily notes
2013-09-28T04:02:26Z
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<br><br />
Notes for July 17th 2013<br />
<br><br />
Activities:<br><br />
1. First briefing with all members of IGEM (advisors, instructors, student) plus some members’s of University’s officials.<br />
<br><br />
2. Self-intoduction from students members about his/her biodata and motivation.<br />
<br><br />
3. Explanation of IGEM Team Sumbawagen schedule, and Human Practice project from Dr. Arief.<br />
<br><br />
4. Election of student leader by students themselves.<br />
<br><br />
<br />
Picture :<br><br />
We are sorry, we have no pictures for the first meeting, because we were too focus for the first meeting.<br />
<br><br />
<a href="http://photozou.jp/photo/show/2832099/188348829"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188348829.png" alt="スクリーンショット 2013-09-28 12.58.18" width="450" height="438"></a><br><a href="http://photozou.jp/photo/show/2832099/188348829"></a> <br><br />
<a href="http://photozou.jp/photo/show/2832099/188348841"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188348841.png" alt="スクリーンショット 2013-09-28 12.58.28" width="450" height="337"></a><br><a href="http://photozou.jp/photo/show/2832099/188348841"></a> <br><br />
<a href="http://photozou.jp/photo/show/2832099/188348848"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188348848.png" alt="スクリーンショット 2013-09-28 12.58.39" width="450" height="181"></a><br><a href="http://photozou.jp/photo/show/2832099/188348848"></a> <br><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Daily_notes
Team:Sumbawagen/Daily notes
2013-09-28T04:01:55Z
<p>Jinhee: </p>
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<br><br />
Notes for July 17th 2013<br />
<br><br />
Activities:<br><br />
1. First briefing with all members of IGEM (advisors, instructors, student) plus some members’s of University’s officials.<br />
<br><br />
2. Self-intoduction from students members about his/her biodata and motivation.<br />
<br><br />
3. Explanation of IGEM Team Sumbawagen schedule, and Human Practice project from Dr. Arief.<br />
<br><br />
4. Election of student leader by students themselves.<br />
<br><br />
<br />
Picture :<br><br />
We are sorry, we have no pictures for the first meeting, because we were too focus for the first meeting.<br />
<br><br />
<a href="http://photozou.jp/photo/show/2832099/188348829"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188348829.png" alt="スクリーンショット 2013-09-28 12.58.18" width="450" height="438"></a><br><a href="http://photozou.jp/photo/show/2832099/188348829">スクリーンショット 2013-09-28 12.58.18</a> <br><br />
<a href="http://photozou.jp/photo/show/2832099/188348841"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188348841.png" alt="スクリーンショット 2013-09-28 12.58.28" width="450" height="337"></a><br><a href="http://photozou.jp/photo/show/2832099/188348841">スクリーンショット 2013-09-28 12.58.28</a> <br><br />
<a href="http://photozou.jp/photo/show/2832099/188348848"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188348848.png" alt="スクリーンショット 2013-09-28 12.58.39" width="450" height="181"></a><br><a href="http://photozou.jp/photo/show/2832099/188348848">スクリーンショット 2013-09-28 12.58.39</a> <br><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Daily_notes
Team:Sumbawagen/Daily notes
2013-09-28T04:01:06Z
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<br><br />
Notes for July 17th 2013<br />
<br><br />
Activities:<br><br />
1. First briefing with all members of IGEM (advisors, instructors, student) plus some members’s of University’s officials.<br />
<br><br />
2. Self-intoduction from students members about his/her biodata and motivation.<br />
<br><br />
3. Explanation of IGEM Team Sumbawagen schedule, and Human Practice project from Dr. Arief.<br />
<br><br />
4. Election of student leader by students themselves.<br />
<br><br />
<br />
Picture :<br><br />
We are sorry, we have no pictures for the first meeting, because we were too focus for the first meeting.<br />
<br><br />
<a href="http://photozou.jp/photo/show/2832099/188348829"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188348829.png" alt="スクリーンショット 2013-09-28 12.58.18" width="450" height="438"></a><br><a href="http://photozou.jp/photo/show/2832099/188348829">スクリーンショット 2013-09-28 12.58.18</a> <br><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Daily_notes
Team:Sumbawagen/Daily notes
2013-09-28T03:57:39Z
<p>Jinhee: </p>
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<br><br />
Notes for July 17th 2013<br />
<br><br />
Activities:<br><br />
1. First briefing with all members of IGEM (advisors, instructors, student) plus some members’s of University’s officials.<br />
<br><br />
2. Self-intoduction from students members about his/her biodata and motivation.<br />
<br><br />
3. Explanation of IGEM Team Sumbawagen schedule, and Human Practice project from Dr. Arief.<br />
<br><br />
4. Election of student leader by students themselves.<br />
<br><br />
<br />
Picture :<br><br />
We are sorry, we have no pictures for the first meeting, because we were too focus for the first meeting.<br />
<br><br />
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Jinhee
http://2013.igem.org/Team:Tokyo-NoKoGen/questionnaire
Team:Tokyo-NoKoGen/questionnaire
2013-09-28T03:50:17Z
<p>Jinhee: </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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<div id="mymenubar"><br />
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<table><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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<td class="project"><a href="https://2013.igem.org/Team:Tokyo-NoKoGen/Project"><span class="project"></span></a></td><br />
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<div id="index"><br />
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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 />
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<div id="main"><br />
<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 />
<p align=center><font size=4> </font></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 width=600></p><br />
<BR><br />
<BR><br />
<p align=center><font size=4>Most of people who know Synthetic biology get the information about synthetic biology from their friends .</font></p><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 />
<p align=center><font size=4>When people are asked “what do you think Synthetic biology is about?”, most of people have right recognition about synthetic biology. </font></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 />
<p align=center><font size=4>About 60% of people don’t know iGEM. </font></p><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 />
<p align=center><font size=4>Most of people who know iGEM, they knew the iGEM from their friend as well as Q5. So, people who know synthetic biology probably know iGEM. </font></p><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 />
<p align=center><font size=4>About 60% people know right pronunciation of iGEM. </font></p><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 />
<p align=center><font size=4>About 50% of people don’t know where iGEM Jamboree held. </font></p><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=700></p><br />
<BR><br />
<p align=center><font size=4>回答は三パターンにわかれた. </font></p><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 />
<p align=center><font size=4>About 50% of people recognize the right number of Japanese teams take part in iGEM. </font></p><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 />
<p align=center><font size=4>Most answered question is the Good for science. But the second is Dirty. People who specialize in science can recognize E.coli was the Good for science. But common people tend to imagine that E.coli is dirty. </font></p><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 />
<p align=center><font size=4>Most answered question is advenced technology but the second is "scary". </font></p><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 />
<p align=center><font size=4>About 70% of people don’t know that modified E.coli can collect heavy metal ions. </font></p><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 />
<p align=center><font size=4>About 70% of people don’t know that modified E.coli can become luminescent. </font></p><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 />
<p align=center><font size=4>About 70% of people don’t know that modified E.coli can can be regulated by light. </font></p><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。そのイカをどうしたでしょう。</font></p><br />
<p align=center><img src=https://static.igem.org/mediawiki/2013/e/e6/Q19.JPG></p><br />
<BR><br />
<p align=center><font size=4>About 50 % of people imagine correctly what we have done with a squid. And this is the most important question because people who know the right answer recognize that we can enjoy synthetic biology using familiar things around everyday life. </font></p><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 />
<p align=center><font size=4>Most of people became interested in synthetic biology via this questionarier. So, we were succeeded to attract people to synthetic biology. </font></p><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 />
<BR><br />
<p align=center><font size=4>As the results of Q20 and Q21, most of people became interested in synthetic biology but about 60% of people don’t feel like to participate iGEM competiting. This is because awareness of iGEM is still low. So when people recognize iGEM is very interesting competition, more people can feel like to participate in iGEM!!. </font></p><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Notebook
Team:Sumbawagen/Notebook
2013-09-28T03:46:18Z
<p>Jinhee: </p>
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<a href="http://photozou.jp/photo/show/2832099/188347227"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347227.png" alt="Fahmi explained about transformation process" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347227">Fahmi explained about transformation process</a> <br />
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<a href="http://photozou.jp/photo/show/2832099/188347234"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347234.png" alt="Honey glucose measurement" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347234">Honey glucose measurement</a><br />
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<a href="http://photozou.jp/photo/show/2832099/188347245"><img src="http://kura3.photozou.jp/pub/99/2832099/photo/188347245.png" alt="Pouring medium into petri dishes" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347245">Pouring medium into petri dishes</a><br />
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<a href="http://photozou.jp/photo/show/2832099/188347255"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347255.jpg" alt="Prof. Sode comments after Sumbawagen iGEM Team presentation" width="450" height="338"></a><br><a href="http://photozou.jp/photo/show/2832099/188347255">Prof. Sode comments after Sumbawagen iGEM Team presentation</a> <br />
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<a href="http://photozou.jp/photo/show/2832099/188347264"><img src="http://kura3.photozou.jp/pub/99/2832099/photo/188347264.png" alt="Study" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347264">Study</a> <br />
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<a href="http://photozou.jp/photo/show/2832099/188347275"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347275.jpg" alt="Sumbawagen team after presentation" width="450" height="338"></a><br><a href="http://photozou.jp/photo/show/2832099/188347275">Sumbawagen team after presentation</a><br />
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<a href="http://photozou.jp/photo/show/2832099/188347289"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347289.png" alt="Sumbawagen team and Prof. Koji Sode after seminar" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347289">Sumbawagen team and Prof. Koji Sode after seminar</a> <br />
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<a href="http://photozou.jp/photo/show/2832099/188347301"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347301.png" alt="Sumbawagen team in lab" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347301">Sumbawagen team in lab</a> <br />
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<a href="http://photozou.jp/photo/show/2832099/188347318"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347318.png" alt="Sumbawagen team surveyed in senior high school" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347318">Sumbawagen team surveyed in senior high school</a><br />
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<a href="http://photozou.jp/photo/show/2832099/188347336"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347336.png" alt="Sumbawagen team with Ms. Dwi" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347336">Sumbawagen team with Ms. Dwi</a> <br />
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<a href="http://photozou.jp/photo/show/2832099/188347346"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347346.png" alt="Sumbawagen team with Prof. Koji Sode and Dr. Tomohiko Yamazaki" width="448" height="302"></a><br><a href="http://photozou.jp/photo/show/2832099/188347346">Sumbawagen team with Prof. Koji Sode and Dr. Tomohiko Yamazaki</a> <br><br />
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<a href="http://photozou.jp/photo/show/2832099/188347370"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347370.png" alt="Sumbawagen team with SUT staffs" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347370">Sumbawagen team with SUT staffs</a><br><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Notebook
Team:Sumbawagen/Notebook
2013-09-28T03:45:15Z
<p>Jinhee: </p>
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<br><br />
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<a href="http://photozou.jp/photo/show/2832099/188347275"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347275.jpg" alt="Sumbawagen team after presentation" width="450" height="338"></a><br><a href="http://photozou.jp/photo/show/2832099/188347275">Sumbawagen team after presentation</a><br />
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<a href="http://photozou.jp/photo/show/2832099/188347318"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347318.png" alt="Sumbawagen team surveyed in senior high school" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347318">Sumbawagen team surveyed in senior high school</a><br />
<br><br />
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<a href="http://photozou.jp/photo/show/2832099/188347336"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347336.png" alt="Sumbawagen team with Ms. Dwi" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347336">Sumbawagen team with Ms. Dwi</a> <br />
<br><br />
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<a href="http://photozou.jp/photo/show/2832099/188347346"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347346.png" alt="Sumbawagen team with Prof. Koji Sode and Dr. Tomohiko Yamazaki" width="448" height="302"></a><br><a href="http://photozou.jp/photo/show/2832099/188347346">Sumbawagen team with Prof. Koji Sode and Dr. Tomohiko Yamazaki</a> <br><br />
<br />
<a href="http://photozou.jp/photo/show/2832099/188347356"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347356.png" alt="Sumbawagen team with SUT staffs-3" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347356">Sumbawagen team with SUT staffs-3</a><br><br />
<br />
<a href="http://photozou.jp/photo/show/2832099/188347370"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347370.png" alt="Sumbawagen team with SUT staffs" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347370">Sumbawagen team with SUT staffs</a><br><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Notebook
Team:Sumbawagen/Notebook
2013-09-28T03:42:59Z
<p>Jinhee: </p>
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<br />
<a href="http://photozou.jp/photo/show/2832099/188347255"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347255.jpg" alt="Prof. Sode comments after Sumbawagen iGEM Team presentation" width="450" height="338"></a><br><a href="http://photozou.jp/photo/show/2832099/188347255">Prof. Sode comments after Sumbawagen iGEM Team presentation</a> <br />
<br><br />
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<a href="http://photozou.jp/photo/show/2832099/188347264"><img src="http://kura3.photozou.jp/pub/99/2832099/photo/188347264.png" alt="Study" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347264">Study</a> <br />
<br><br />
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<a href="http://photozou.jp/photo/show/2832099/188347275"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347275.jpg" alt="Sumbawagen team after presentation" width="450" height="338"></a><br><a href="http://photozou.jp/photo/show/2832099/188347275">Sumbawagen team after presentation</a><br />
<br><br />
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<a href="http://photozou.jp/photo/show/2832099/188347289"><img src="http://kura1.photozou.jp/pub/99/2832099/photo/188347289.png" alt="Sumbawagen team and Prof. Koji Sode after seminar" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347289">Sumbawagen team and Prof. Koji Sode after seminar</a> <br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Notebook
Team:Sumbawagen/Notebook
2013-09-28T03:39:46Z
<p>Jinhee: </p>
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<a href="http://photozou.jp/photo/show/2832099/188347227"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347227.png" alt="Fahmi explained about transformation process-3" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347227">Fahmi explained about transformation process-3</a> <br />
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<a href="http://photozou.jp/photo/show/2832099/188347234"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347234.png" alt="Honey glucose measurement" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347234">Honey glucose measurement</a><br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Notebook
Team:Sumbawagen/Notebook
2013-09-28T03:39:16Z
<p>Jinhee: </p>
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#footer-box {<br />
width: 1410px;<br />
height: ;<br />
}<br />
<br />
#footer {<br />
border: none;<br />
width: 1410px;<br />
}<br />
<br />
.firstHeading {<br />
display: none;<br />
}<br />
<br />
#f-list a {<br />
color: #333;<br />
font-size: 10px;<br />
}<br />
<br />
#f-list a:hover {<br />
color: #666;<br />
}<br />
<br />
.printfooter {<br />
display: none;<br />
}<br />
<br />
h3#siteSub {<br />
display: none;<br />
}<br />
<br />
#contentSub {<br />
display: none;<br />
}<br />
<br />
<br />
#menu{ <br />
<br />
width:400px;<br />
<br />
}<br />
<br />
<br />
<br />
</style><br />
<br />
<br />
</head><br />
<body style="background: rgb(255, 255, 255) url(https://static.igem.org/mediawiki/2013/4/4b/Playtime_background_yellow.jpg) repeat scroll top left; margin: 0pt; " onload="onPageLoad();" onunload="onPageUnload();"><br />
<div style="text-align: center; "><br />
<div style="margin-bottom: 15px; margin-left: auto; margin-right: auto; margin-top: 15px; overflow: hidden; position: relative; word-wrap: break-word; text-align: left; width: 700px; " id="body_content"><br />
<div style="background: transparent url(https://static.igem.org/mediawiki/2013/6/65/Playtime_page_bg-6.png) repeat scroll top left; width: 700px; "><br />
<div style="height: 111px; margin-left: 0px; position: relative; width: 700px; z-index: 10; " id="header_layer"><br />
<div style="height: 0px; line-height: 0px; " class="bumper"> </div><br />
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<div class="text-content style_External_645_61" style="padding: 0px; "><br />
<div class="style"><br />
<p style="padding-bottom: 0pt; padding-top: 0pt; " class="paragraph_style">Sumbawagen iGEM Team</p><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<br />
<div style="height: 22px; width: 645px; height: 22px; left: 22px; position: absolute; top: 29px; width: 645px; z-index: 1; " class="tinyText style_SkipStroke_1"><br />
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<img src="https://static.igem.org/mediawiki/2013/4/40/Kids_green_header_02_1.png" alt="" style="border: none; height: 86px; width: 645px; " /><br />
</div><br />
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<div style="height: 20px; width: 700px; height: 20px; left: 0px; position: absolute; top: 0px; width: 700px; z-index: 1; " class="tinyText style_SkipStroke_1"><br />
<img src="https://static.igem.org/mediawiki/2013/3/36/Playtime_header_bg.jpg" alt="" style="border: none; height: 20px; width: 700px; " /><br />
</div><br />
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<div style="height: 172px; width: 190px; height: 172px; left: 495px; position: absolute; top: 15px; width: 190px; z-index: 1; " class="tinyText style_SkipStroke_1"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/Playtime_Sun-4.png" alt="" style="border: none; height: 172px; width: 190px; " /><br />
</div><br />
<div style="height: 80px; width: 80px; height: 80px; left: 10px; position: absolute; top: 15px; width: 190px; z-index: 1; " class="tinyText style_SkipStroke_1"><br />
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</a></div><br />
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</div><br />
<div style="margin-left: 0px; position: relative; width: 700px; z-index: 0; " id="nav_layer"><br />
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<div style="height: 1px; line-height: 1px; " class="tinyText"> </div><br />
<div class="com-apple-iweb-widget-navbar flowDefining" id="widget0" style="margin-left: 22px; margin-top: -1px; opacity: 1.00; position: relative; width: 645px; z-index: 1; "><br />
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<div id="widget0-navbar" class="navbar"><br />
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<div id="widget0-bg" class="navbar-bg"><br />
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<ul id="widget0-navbar-list" class="navbar-list"><br />
<li></li> <br />
</ul><br />
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</div><br />
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<div id="menu"><br />
<A Href="https://2013.igem.org/Team:Sumbawagen">Home</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Team">Team</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Modeling">Modeling</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Project">Project</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Project">Parts</A><br />
Notebook<br />
<br><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Lab Preparation team activities">Lab Preparation team activities</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Safety">Safety</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Acknowledgement">Acknowledgement</A><br />
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<div style="clear: both; height: 0px; line-height: 0px; " class="spacer"> </div><br />
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<div style="margin-left: 0px; position: relative; width: 700px; z-index: 5; " id="body_layer"><br />
<div style="height: 0px; line-height: 0px; " class="bumper"> </div><br />
<div id="id2" style="height: 34px; left: 35px; position: absolute; top: 17px; width: 620px; z-index: 1; " class="style_SkipStroke_2 shape-with-text"><br />
<div class="text-content Normal_External_620_34" style="padding: 0px; "><br />
<div class="Normal"><br />
<br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Protocols">Protocols</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Artwork_pictures">Artwork pictures</A><br />
<A Href="https://2013.igem.org/Team:Sumbawagen/Daily_notes">Daily notes</A><br />
</div><br />
</div><br />
</div><br />
<br />
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<div style="height: 16px; width: 645px; height: 16px; left: 22px; position: absolute; top: 0px; width: 645px; z-index: 1; " class="tinyText style_SkipStroke_1"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3a/Kids_green_header_03.png" alt="" style="border: none; height: 16px; width: 645px; " /><br />
</div><br />
<br />
<br />
<br />
<div id="id3" style="height: 2766px; left: 35px; position: absolute; top: 47px; width: 628px; z-index: 1; " class="style_SkipStroke_3 shape-with-text"><br />
<div class="text-content graphic_textbox_layout_style_default_External_628_2766" style="padding: 0px; "><br />
<div class="graphic_textbox_layout_style_default"><br />
<br />
<br><br />
<br><br />
<br />
<a href="http://photozou.jp/photo/show/2832099/188347227"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347227.png" alt="Fahmi explained about transformation process-3" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347227">Fahmi explained about transformation process-3</a> <br />
<br />
<br><br />
<br />
<a href="http://photozou.jp/photo/show/2832099/188347234"><img src="http://kura2.photozou.jp/pub/99/2832099/photo/188347234.png" alt="Honey glucose measurement" width="448" height="336"></a><br><a href="http://photozou.jp/photo/show/2832099/188347234">Honey glucose measurement</a><br />
</div><br />
<div style="clear: both; height: 0px; line-height: 0px; " class="tinyText"> </div><br />
</div><br />
</div><br />
<div style="height: 400px; line-height: 400px; " class="spacer"> </div><br />
</div><br />
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<div style="height: 0px; line-height: 0px; " class="bumper"> </div><br />
<br />
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</a><br />
<div style="height: 17px; width: 700px; height: 17px; left: 0px; position: absolute; top: 58px; width: 700px; z-index: 1; " class="tinyText style_SkipStroke_1"><br />
<img src="https://static.igem.org/mediawiki/2013/b/be/Playtime_footer_bg.jpg" alt="" style="border: none; height: 17px; width: 700px; " /><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</body><br />
</html></div>
Jinhee
http://2013.igem.org/Team:Tokyo-NoKoGen/oscillator
Team:Tokyo-NoKoGen/oscillator
2013-09-28T03:28:24Z
<p>Jinhee: </p>
<hr />
<div><html><br />
<br />
<head><br />
<br />
<style type ='text/css'><br />
<br />
#globalWrapper { <br />
width: 1410px; <br />
height:100%;<br />
}<br />
<br />
#content { <br />
background-color: transparent; <br />
border: none; <br />
padding: 0;<br />
margin: 0; <br />
margin-top: -23px; <br />
width: 100%; <br />
overflow: hidden; <br />
height:100%;<br />
}<br />
<br />
#bodyContent { <br />
border: none; <br />
padding:0; <br />
margin:0; <br />
width:1410px; <br />
height:100%;<br />
}<br />
<br />
#top-section {<br />
height: 18px; <br />
margin: 0px; <br />
margin-top: 5px; <br />
margin-left: auto; <br />
margin-right: auto; <br />
margin-bottom: 0 !important;<br />
padding:0; <br />
border: none; <br />
font-size: 10px;<br />
}<br />
<br />
#p-logo { <br />
overflow:hidden; <br />
display: none;<br />
}<br />
<br />
#search-controls { <br />
display:none; <br />
}<br />
<br />
#footer-box {<br />
width: 1410px; <br />
height: ;<br />
}<br />
<br />
#footer { <br />
border: none; <br />
width: 1410px; <br />
}<br />
<br />
.firstHeading { <br />
display: none;<br />
}<br />
<br />
#f-list a { <br />
color: #333; <br />
font-size: 10px;<br />
}<br />
<br />
#f-list a:hover { <br />
color: #666;<br />
}<br />
<br />
.printfooter { <br />
display: none; <br />
}<br />
<br />
h3#siteSub { <br />
display: none;<br />
}<br />
<br />
#contentSub {<br />
display: none;<br />
}<br />
<br />
p:first-child { <br />
display: none;<br />
}<br />
<br />
h1,h2,h3 {<br />
border: none;<br />
}<br />
<br />
#wrapper {<br />
font-size: 12pt;<br />
font-family: "Comic Sans MS","MS明朝";<br />
width:1410px;<br />
height:100%;<br />
margin:auto ;<br />
}<br />
<br />
#title_logo {<br />
float: left;<br />
width:705px;<br />
height:250px;<br />
background :url("https://static.igem.org/mediawiki/2013/d/d6/Header_index_kiso5.png") ;<br />
}<br />
<br />
#title_logo img.iGEM {<br />
position: relative;<br />
top: 50px;<br />
left: 40px;<br />
width: 150px;<br />
height: auto;<br />
}<br />
<br />
#title_logo img.title {<br />
position: relative;<br />
top:20px;<br />
left:40px;<br />
width:600px;<br />
height:200px;<br />
}<br />
<br />
#mymenubar {<br />
float: left;<br />
width: 705px;<br />
height: 250px;<br />
background: url("https://static.igem.org/mediawiki/2013/d/d6/Header_index_kiso5.png") -705px 0px no-repeat;<br />
}<br />
<br />
#mymenubar table,tbody,tr {<br />
background: transparent;<br />
}<br />
<br />
#mymenubar table td div.home,home1 {<br />
width: 70px;<br />
height: 95px;<br />
}<br />
<br />
#mymenubar table td span.home {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 90px 20px 0px 20px;<br />
background: url("https://static.igem.org/mediawiki/2013/9/96/Home28.png") no-repeat 0px 0px;<br />
}<br />
<br />
#mymenubar table td span.home:hover {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 90px 20px 0px 20px;<br />
background-position: 0px -99px;<br />
}<br />
<br />
<br />
#mymenubar table div.home1 {<br />
width: 70px;<br />
height: 95px;<br />
margin: 92px 20px 0px 20px ;<br />
background: url("https://static.igem.org/mediawiki/2013/1/18/Home26.png") no-repeat 0px 0px ;<br />
background-size: 70px 95px;<br />
}<br />
<br />
#mymenubar table td span.project {<br />
display: block;<br />
width: 70px;<br />
height: 95px;<br />
}<br />
<br />
#mymenubar table td.project {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 88px 20px 0px 0px;<br />
background: url("https://static.igem.org/mediawiki/2013/f/f5/Project28.png") no-repeat 0px 0px;<br />
}<br />
<br />
#mymenubar table td.project:hover {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 88px 20px 0px 0px;<br />
background-position: 0px -99px;<br />
}<br />
<br />
#mymenubar table div.project1 {<br />
width: 70px;<br />
height: 95px;<br />
margin: 92px 20px 0px 0px ;<br />
background: url("https://static.igem.org/mediawiki/2013/f/f9/Project26.png") no-repeat 0px 0px ;<br />
background-size: 70px 95px;<br />
}<br />
<br />
<br />
#mymenubar table td span.team {<br />
display: block;<br />
width: 70px;<br />
height: 95px;<br />
}<br />
<br />
#mymenubar table td.team {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 91px 20px 0px 0px;<br />
background: url("https://static.igem.org/mediawiki/2013/b/ba/Team28.png") no-repeat 0px 0px;<br />
}<br />
<br />
#mymenubar table td.team:hover {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 91px 20px 0px 0px;<br />
background-position: 0px -98px;<br />
}<br />
<br />
#mymenubar table div.team1 {<br />
width: 70px;<br />
height: 95px;<br />
margin: 92px 20px 0px 0px ;<br />
background: url("https://static.igem.org/mediawiki/2013/8/8e/Team26.png") no-repeat 0px 0px ;<br />
background-size: 70px 95px;<br />
}<br />
<br />
<br />
#mymenubar table td span.biobrick {<br />
display: block;<br />
width: 70px;<br />
height: 95px;<br />
}<br />
<br />
#mymenubar table td.biobrick {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 93px 20px 0px -2px;<br />
background: url("https://static.igem.org/mediawiki/2013/9/96/Biobrick28.png") no-repeat 0px 0px;<br />
}<br />
<br />
#mymenubar table td.biobrick:hover {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 93px 20px 0px -2px;<br />
background-position: 0px -99px;<br />
}<br />
<br />
#mymenubar table div.biobrick1 {<br />
width: 70px;<br />
height: 95px;<br />
margin: 92px 20px 0px -2px ;<br />
background: url("https://static.igem.org/mediawiki/2013/c/c9/Biobrick26.png") no-repeat 0px 0px ;<br />
background-size: 70px 95px;<br />
}<br />
<br />
<br />
#mymenubar table td span.notebook {<br />
display: block;<br />
width: 70px;<br />
height: 95px;<br />
}<br />
<br />
#mymenubar table td.notebook {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 95px 20px 0px 0px;<br />
background: url("https://static.igem.org/mediawiki/2013/7/78/Notebook28.png") no-repeat 0px 0px;<br />
}<br />
<br />
#mymenubar table td.notebook:hover {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 95px 20px 0px 0px;<br />
background-position: 0px -100px;<br />
}<br />
<br />
#mymenubar table div.notebook1 {<br />
width: 70px;<br />
height: 95px;<br />
margin: 92px 20px 0px 0px ;<br />
background: url("https://static.igem.org/mediawiki/2013/8/8d/Notebook26.png") no-repeat 0px 0px ;<br />
background-size: 70px 95px;<br />
}<br />
<br />
#mymenubar table td span.humanpractice {<br />
display: block;<br />
width: 70px;<br />
height: 97px;<br />
}<br />
<br />
#mymenubar table td.humanpractice {<br />
float: left;<br />
width: 70px;<br />
height: 97px;<br />
margin: 86px 20px 0px -1px;<br />
background: url("https://static.igem.org/mediawiki/2013/c/c1/Humanpractice28.png") no-repeat 0px 0px;<br />
}<br />
<br />
#mymenubar table td.humanpractice:hover {<br />
float: left;<br />
width: 70px;<br />
height: 97px;<br />
margin: 86px 20px 0px -1px;<br />
background-position: 0px -99px;<br />
}<br />
<br />
#mymenubar table div.humanpractice1 {<br />
width: 70px;<br />
height: 95px;<br />
margin: 92px 20px 0px -1px ;<br />
background: url("https://static.igem.org/mediawiki/2013/5/54/Humanpractice26.png") no-repeat 0px 0px ;<br />
background-size: 70px 95px;<br />
}<br />
<br />
#mymenubar table td span.achievement {<br />
display: block;<br />
width: 70px;<br />
height: 95px;<br />
}<br />
<br />
#mymenubar table td.achievement {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 93px 20px 0px -2px;<br />
background: url("https://static.igem.org/mediawiki/2013/5/5e/Achievement28.png") no-repeat 0px 0px;<br />
}<br />
<br />
#mymenubar table td.achievement:hover {<br />
float: left;<br />
width: 70px;<br />
height: 95px;<br />
margin: 93px 20px 0px -2px;<br />
background-position: 0px -99px;<br />
}<br />
<br />
#mymenubar table div.achievement1 {<br />
width: 70px;<br />
height: 95px;<br />
margin: 92px 20px 0px -2px ;<br />
background: url("https://static.igem.org/mediawiki/2013/5/5a/Achievement26.png") no-repeat 0px 0px ;<br />
background-size: 70px 95px;<br />
}<br />
<br />
#index {<br />
float: left;<br />
width:490px;<br />
height:8000px;<br />
background: url("https://static.igem.org/mediawiki/2013/d/d6/Header_index_kiso5.png") 0px -250px no-repeat ;<br />
}<br />
<br />
<br />
#index img.logo {<br />
position: relative;<br />
top:20px;<br />
left:230px;<br />
width:150px;<br />
height:150px;<br />
}<br />
<br />
#index_title {<br />
position: relative;<br />
top:40px;<br />
left:230px;<br />
}<br />
<br />
#contents {<br />
position: relative;<br />
width:100px;<br />
top:240px;<br />
left:220px;<br />
}<br />
<br />
#contents2 {<br />
position: relative;<br />
width: 100px;<br />
top: 340px;<br />
left: 220px;<br />
}<br />
<br />
#main {<br />
float: left;<br />
width:820px;<br />
height:8000px;<br />
background: url("https://static.igem.org/mediawiki/2013/d/d6/Header_index_kiso5.png") -490px -250px no-repeat;<br />
<br />
}<br />
<br />
#space {<br />
float: left;<br />
width: 100px;<br />
height: 8000px;<br />
background: url("https://static.igem.org/mediawiki/2013/d/d6/Header_index_kiso5.png") -1310px -250px no-repeat;<br />
}<br />
<br />
#footerr {<br />
clear: both;<br />
width: 1410px;<br />
height: 700px;<br />
background: url("https://static.igem.org/mediawiki/2013/d/d6/Header_index_kiso5.png") 0px -9300px no-repeat;<br />
}<br />
<br />
#footer_sponsors ul {<br />
margin-left: 300px;<br />
}<br />
<br />
#footer_sponsors li {<br />
float: left;<br />
list-style-type: none;<br />
height: 70px;<br />
}<br />
<br />
#footer_sponsors li.UI {<br />
width:150px;<br />
margin: 50px 0px 0px 370px;<br />
}<br />
<br />
#footer_sponsors li.CB {<br />
width: ;<br />
margin: 80px 0px 0px 50px;<br />
}<br />
<br />
#footer_sponsors li.LN {<br />
width:;<br />
margin: 50px 0px 0px 50px;<br />
}<br />
<br />
#footer_sponsors li.IR {<br />
clear: both;<br />
width:;<br />
margin: 45px 0px 0px 370px;<br />
}<br />
<br />
#footer_sponsors li.Promega {<br />
width:;<br />
margin: 30px 0px 0px 50px;<br />
}<br />
<br />
#footer_sponsors li.IDT {<br />
width:;<br />
margin: 55px 0px 0px 50px;<br />
}<br />
<br />
#footer_sponsors li.MBL {<br />
width:;<br />
margin: 50px 0px 0px 370px;<br />
}<br />
<br />
#footer_sponsors li.NK {<br />
clear: both;<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="#TR-HHR">TR-HHR</a></li><br />
<li><a href="#TR-HHR-taRNA">TR-HHR-taRNA</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 />
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<div id="main"><br />
<br />
<font size=5><br />
<BR><br />
<p align=center><font size=7><strong>RNA Oscillator</strong></font><hr></p><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6><strong>Introduction </span></strong></font><hr><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6 ><strong>Protein oscillator</span></strong></font><br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
(There are many kinds of gene regulation system. One of them is repressor system that is used...)<br />
<BR><br />
The oscillating network was built by using three transcriptional repressor systems in which the first repressor protein suppresses the expression of second repressor gene, whose protein suppresses the transcription of the third gene, and the protein the third gene code suppresses the first gene expression.<br />
</p><br />
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<BR><br />
<BR><br />
<br />
<font size=6><strong>Hummer headed ribozyme</span></strong></font><br />
<br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
Hummerheaded ribozymes (HHRs) are found in many species and known to have a capability to self-cleave. Recently, engineered HHRs responding to trans-acting RNAs (taRNAs) in <I>E.coli</I> were reported. In absence of taRNA, the HHRs can self-cleave, but in presence of taRNA, their capability of self-cleavage is inhibited due to hybridize with taRNA and change their structures.<br />
</p><br />
<BR><br />
<img src=https://static.igem.org/mediawiki/2013/3/3a/%E3%82%AD%E3%83%A3%E3%83%97%E3%83%81%E3%83%A318.PNG><BR><br />
Fig. 1 taRNAs responsible HHR<br />
<BR><BR><BR><br />
<br />
<p style="line-height:110%"><br />
We developed the new oscillator system that is composed only RNAs not using repressor gene and protein. We created three taRNA connected hummerheaded ribozymes (HHR-taRNA) that sense to another taRNA. The same way as the protein oscillator, three HHR-taRNAs inhibits other one’s self-cleavage such as three-way standoff. When the first HHR-taRNA is self-cleaved, connected the first taRNA that binds to the second HHR-taRNA is free and suppresses the second HHR-taRNA self-cleavage. When second HHR-taRNA is self-cleaved, the second taRNA that binds to the third HHR-taRNA is free and suppresses the third HHR-taRNA. Finally, when third HHR-taRNA is self-cleaved, the third taRNA that binds to the first HHR-taRNA is free and suppresses the first HHR-taRNA, completing cycle.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
<font size=6 id="Objective"><strong>Objective</span></strong></font><hr> <br />
<BR><br />
<BR><br />
Construction of Three of orthogonal taRNA generator which repressed by another taRNA.<br />
<br />
<BR><br />
<br />
<p style="line-height:110%"><br />
taRNA responsive HHR (TR-HHR) was fused with taRNA. By using orthogonal riboregulator sequences, we designed three of taRNA generators.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
<font size=6 id="Method"><strong>Method</span></strong></font><hr><br />
<br />
<BR><BR><br />
<br />
<font size=5 id="Design"><strong>Design</span></strong></font> <br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
TR-HHR was designed by Benedikt Klauser and Jo¨ rg S. Hartig. Their TR-HHR were inspired by the riboregulatory system previously reported by Collins and coworkers. They made use of sequence domains of the riboregulator RR12. And, orthogonal variants of riboregulator RR12 (RR12y, RR42) were reported by Collins and coworkers. So, by using the sequences of these orthogonal riboregulators, we designed three orthogonal TR-HHRs.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
図1<br />
<br />
<BR><BR><BR><br />
<br />
<font size=5 id="Parts construction"><strong>Parts construction</span></strong></font> <br />
<BR><br />
<p style="line-height:110%"><br />
1. Construction of TR-HHR<BR><br />
2. Construction of TR-HHR variants<BR><br />
3. Construction of taRNA variants<BR><br />
4. Construction of TR-HHR-taRNA<BR><br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
1. Construction of TR-HHR<BR><br />
<br />
<p style="line-height:110%"> <br />
We used the TR-HHR developed by Klauser <I>et al.</I>[1] Two overlap primers designed, and PCR was performed to connect them. The PCR product was ligated into pSB1C3 vector.<br />
</p><br />
<br />
<BR><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e3/Wiki1.png" width="400px" / ><br />
<br />
<BR><BR><br />
<br />
2.Construction of TR-HHR variants<br />
<br />
<BR><BR><BR><br />
<br />
<p style="line-height:110%"><br />
By inverse PCR using pSB1C3-TR-HHR as a template, we could obtain three TR-HHRs, TR(12)-HHR, TR(12y)-HHR, and TR(42)-HHR.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/9/98/Wiki6.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<br />
<br />
3.Construction of taRNA variants<br />
<br />
<BR><br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
By inverse PCR using pSB1A3-taR12-DT as a template, we could obtain three taRNAs, taR12, taR12y, taR42.<br />
</p><br />
<br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a7/Wiki2.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<br />
4.Construction of TR-HHR-taRNA<br />
<BR><br />
<BR><br />
<BR><br />
<br />
TR-HHR-taRNAs were constructed by overlap PCR.<br />
<br />
<BR><br />
<BR><br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/9/91/Wiki3.png" width="400px" / ><br />
<br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<BR><br />
<font size=6 id="Evaluation"><strong>Evaluation</strong></font><hr><br />
<BR><br />
<BR><br />
I. Evaluation of TR-HHR<br />
<BR><BR><br />
II. Evaluation of TR-HHR-taRNA<br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="TR-HHR"><strong>I. Evaluation of TR-HHR</strong></font><br />
<br />
<BR><br />
<br />
<p style="line-height:110%"><br />
In order to evaluate the regulation of HHR’s self-cleavage activity by taRNA, GFP gene was connected with the HHR or inactive HHR (*HHR), which has a mutation lose the activity. These fragments were subcloned into conventional biobrick standard vector pSB3C5. <br />
</p><br />
<br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/4/45/Wiki4.png" width="400px" / ><br />
<BR><br />
<br />
1) <I>E. coli</I> TOP10 strain was co-transformed by two plasmids listed bellow, <br />
<BR><br />
<BR><br />
A)<br />
<BR><br />
pSB1A3-taR12-DT<br />
<BR><br />
pSB3C5-TR(12)-HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
B)<br />
<BR><br />
pSB1A3-empty<br />
<BR><br />
pSB3C5- TR(12)-HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
C) <br />
<BR><br />
pSB1A3-taR12-DT<br />
<BR><br />
pSB3C5-TR(12)- * HHR-GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
D)<br />
<BR><br />
pSB1A3 empty<br />
<BR><br />
pSB3C5- TR(12)- * HHR -GFP-DT<br />
<br />
<BR><br />
<BR><br />
<br />
<br />
E) <br />
<BR>pSB1A3-empty<br />
<BR><br />
pSB3C5-empty<br />
<br />
<BR><br />
<BR><br />
<br />
<p style="line-height:110%"><br />
2) Bacterial cultures were incubated in LB medium at 140 rpm and 37℃ for 12 hour. <BR><br />
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.<BR><br />
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.<BR><br />
5) OD595 and GFP fluorescence intensity were measured.<BR><br />
All experiments were performed using three cultures per each sample.<br />
</p><br />
<br />
<BR><BR><BR><br />
<br />
Result and Discussion<br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
In the presence of taR12, GFP fluorescence value divided by OD value is higher than one in absence of taR12. This result was not expected. HHR has the ability to self-cleave, but its ability is suppressed when taRNA hybridizes to HHR. Therefore, in the presence of taRNA, self-cleavage of HHR should not happen. So then, why we get such result? One factor can be due to the exposure of RBS sequence caused by the structural change of HHR when it hybridizes to taRNA<br />
</p><br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="TR-HHR-taRNA">II.Evaluation of TR-HHR-taRNA</font><br />
<br />
<BR><BR><br />
<br />
We examined whether TR-HHR-taRNA’s output can be repressed by corresponding taRNA. <br />
<BR><br />
<BR><br />
<img src="https://static.igem.org/mediawiki/igem.org/5/52/Wiki5.png" width="400px" / ><br />
<BR><br />
<br />
<br />
1) The plasmids as shown below was used to transform <I>E. coli </I>Top10.<BR><br />
<BR><br />
A) <br />
<BR><br />
pSB1A3-P<sub>bad</sub> -TR(42)HHR-taR12-DT<br />
<BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
B) <br />
<BR><br />
pSB1A3-P<sub>const(H)</sub>-taR42-P<sub>bad</sub>-TR(42)HHR-taR12-DT<BR><br />
pSB3C5-crR12-GFP-DT<br />
<br />
<BR><BR><BR><br />
<br />
C)<br />
<BR><br />
pSB1A3-P<sub>bad</sub>-taR12-DT<BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
D) <BR><br />
pSB1A3-empty <BR><br />
pSB3C5-crR12-GFP<br />
<br />
<BR><BR><BR><br />
<br />
E) <BR><br />
pSB1A3-empty<BR><br />
pSB3C5-empty<BR><br />
<br />
<BR><BR><br />
<br />
<p style="line-height:110%"><br />
2) The transformants were pre-cultured in LB medium at 37 degrees celcius for 8 hours. <BR><br />
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.<BR><br />
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.<BR><br />
5) OD595 and GFP fluorescence intensity were measured.<BR><br />
</p><br />
<br />
<BR><BR><br />
<br />
<font size=5 ><strong>Result and Discussion</strong></font><hr><br />
<br />
<BR><br />
<BR><br />
<p style="line-height:110%"><br />
In order to evaluate a function of TR(42)-HHR-taR12, we used crR12-GFP as a reporter.<br />
taRNA is complementary to the sequence of crRNA, and by the binding of taRNA to crRNA, RBS is exposed and gene expression is activated. In this result, the GFP fluorescence value divided by OD value of (2) was lower than that of (3). It shows that taR42 suppressed self-cleavage of TR(42)-HHR and caused less GFP expression. Therefore, we prove that the self-cleavage activity of TR(42)-HHR and amount of taR12 can be controlled by taR42.<br />
</p><br />
<br />
<BR><BR><BR><BR><br />
<br />
<font size=6 id="Future work" ><strong>・Future work</strong></font><hr><br />
<br />
<BR><BR><BR><BR><BR><br />
<br />
<font size=6 ><strong>・Reference</strong></font><br />
<br />
<BR><BR><br />
<BR><br />
<p style="line-height:110%"><br />
[1]<br />
An engineered small RNA-mediated genetic switch<br />
based on a ribozyme expression platform<br />
Benedikt Klauser and Jo¨ rg S. Hartig <br />
Nucleic Acids Research, 2013<br />
<BR><br />
[2]<br />
Callura,J.M., Cantor,C.R. and Collins,J.J. (2012) Genetic<br />
switchboard for synthetic biology applications. Proc. Natl Acad.<br />
Sci. USA, 109, 5850–5855<br />
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Daily_notes
Team:Sumbawagen/Daily notes
2013-09-28T02:41:12Z
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Lab_Preparation_team_activities
Team:Sumbawagen/Lab Preparation team activities
2013-09-28T02:40:13Z
<p>Jinhee: </p>
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Lab_Preparation_team_activities
Team:Sumbawagen/Lab Preparation team activities
2013-09-28T02:39:35Z
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Jinhee
http://2013.igem.org/File:%E3%82%B9%E3%82%AF%E3%83%AA%E3%83%BC%E3%83%B3%E3%82%B7%E3%83%A7%E3%83%83%E3%83%88_2013-09-28_11.21.47.png
File:スクリーンショット 2013-09-28 11.21.47.png
2013-09-28T02:25:31Z
<p>Jinhee: </p>
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Jinhee
http://2013.igem.org/Team:Sumbawagen/Lab_Preparation_team_activities
Team:Sumbawagen/Lab Preparation team activities
2013-09-28T02:20:07Z
<p>Jinhee: </p>
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Jinhee
http://2013.igem.org/File:L6.png
File:L6.png
2013-09-28T02:19:32Z
<p>Jinhee: </p>
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Jinhee
http://2013.igem.org/File:L5.png
File:L5.png
2013-09-28T02:16:03Z
<p>Jinhee: </p>
<hr />
<div></div>
Jinhee
http://2013.igem.org/Team:Sumbawagen/Lab_Preparation_team_activities
Team:Sumbawagen/Lab Preparation team activities
2013-09-28T02:13:53Z
<p>Jinhee: </p>
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Jinhee