http://2013.igem.org/wiki/index.php?title=Special:Contributions/Niina&feed=atom&limit=50&target=Niina&year=&month=2013.igem.org - User contributions [en]2024-03-28T16:28:55ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T03:54:38Z<p>Niina: /* RT-PCR */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what makes this difference between dry work and wet work, and makes modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment, the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero. Consequently, this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is adoption of robust gene-circuit model in order to ignore the complexity by approximation. However, there are difficulties in choosing factors under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is generating oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation becomes narrower with RNA that is produced or discomposed speedy, we think. We choose Spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: Before starting the oscillation, this circuit doesn't generate oscillation due to the repression of attenuator-TetR aptamer by lacI. First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because TetR aptamer activates Ptet, positive feedback occurs and more and more TetR aptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, TetR aptamer, at the downstream of Attenuator region, is repressed. Then, because new TetR aptamer is not created, the amount of TetR aptamer decreases quickly. Therefore, Ptet is repressed by TetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allows transcription of the downstream (Novick et al, 1989)[6]. The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists vary functions of RNA only by means of nucleotide substitution etc. (Takahashi et al, 2013)[2].<br />
In this paper, many variants of pT181 attenuator/antisense are constructed and the attenuation rate of each variant is different. We chose this mechanism for gene repression. 2013IGKUprojectRNArepressionMECHANISM.png<br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding with tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, it induces the conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix.We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay.<br />
[[File:No-binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it manifests that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also to reflect the transcription level accurately, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
[[File:SPINACHの説明.png]]<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we constructed functional RNA generator, we checked the transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Non-promoter: Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also checked whether fusion RNA we designed functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of TetR aptamer(left) and Spinach(center).<br><br />
[[File:ElectrophoresisRT1.png]]<br />
[[File:ElectrophoresisRT2.png]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-TetRaptamer and antisence-Spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between TetR and TetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay TatR aptamer<br><br />
To confirm the act of TetR aptamer inducing Ptet ,we allow IPTG-inducble TetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, Spinach, no-RNA and attenuator-TetR aptamer. As positive controls, we also use GFP.<br><br />
3, qualitatively Spinach assay (visual recognition & fluorescence microscopes)<br><br />
We check that DFHBI fluorescence on a plate with Spinach.<br><br />
We cultivate IPTG-inducible Spinach in a liquid culture under a shading condition, and add DFHBI. Then we check whether this sample fluorescence after centrifugation. We also check Spinach-GFP and antisense-Spinach.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br><br />
[6][http://www.ncbi.nlm.nih.gov/pubmed/2478296 Novick RP et al. (1999) "pT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator"]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T03:54:07Z<p>Niina: /* RT-PCR */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what makes this difference between dry work and wet work, and makes modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment, the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero. Consequently, this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is adoption of robust gene-circuit model in order to ignore the complexity by approximation. However, there are difficulties in choosing factors under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is generating oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation becomes narrower with RNA that is produced or discomposed speedy, we think. We choose Spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: Before starting the oscillation, this circuit doesn't generate oscillation due to the repression of attenuator-TetR aptamer by lacI. First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because TetR aptamer activates Ptet, positive feedback occurs and more and more TetR aptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, TetR aptamer, at the downstream of Attenuator region, is repressed. Then, because new TetR aptamer is not created, the amount of TetR aptamer decreases quickly. Therefore, Ptet is repressed by TetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allows transcription of the downstream (Novick et al, 1989)[6]. The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists vary functions of RNA only by means of nucleotide substitution etc. (Takahashi et al, 2013)[2].<br />
In this paper, many variants of pT181 attenuator/antisense are constructed and the attenuation rate of each variant is different. We chose this mechanism for gene repression. 2013IGKUprojectRNArepressionMECHANISM.png<br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding with tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, it induces the conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix.We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay.<br />
[[File:No-binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it manifests that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also to reflect the transcription level accurately, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
[[File:SPINACHの説明.png]]<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we constructed functional RNA generator, we checked the transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Non-promoter: Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also checked whether fusion RNA we designed functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of TetR aptamer(center) and Spinach(left).<br><br />
[[File:ElectrophoresisRT1.png]]<br />
[[File:ElectrophoresisRT2.png]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-TetRaptamer and antisence-Spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between TetR and TetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay TatR aptamer<br><br />
To confirm the act of TetR aptamer inducing Ptet ,we allow IPTG-inducble TetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, Spinach, no-RNA and attenuator-TetR aptamer. As positive controls, we also use GFP.<br><br />
3, qualitatively Spinach assay (visual recognition & fluorescence microscopes)<br><br />
We check that DFHBI fluorescence on a plate with Spinach.<br><br />
We cultivate IPTG-inducible Spinach in a liquid culture under a shading condition, and add DFHBI. Then we check whether this sample fluorescence after centrifugation. We also check Spinach-GFP and antisense-Spinach.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br><br />
[6][http://www.ncbi.nlm.nih.gov/pubmed/2478296 Novick RP et al. (1999) "pT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator"]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/File:ElectrophoresisRT2.pngFile:ElectrophoresisRT2.png2013-09-28T03:51:51Z<p>Niina: </p>
<hr />
<div></div>Niinahttp://2013.igem.org/File:ElectrophoresisRT1.pngFile:ElectrophoresisRT1.png2013-09-28T03:49:45Z<p>Niina: </p>
<hr />
<div></div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T03:47:55Z<p>Niina: /* RT-PCR */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what makes this difference between dry work and wet work, and makes modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment, the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero. Consequently, this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is adoption of robust gene-circuit model in order to ignore the complexity by approximation. However, there are difficulties in choosing factors under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is generating oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation becomes narrower with RNA that is produced or discomposed speedy, we think. We choose Spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: Before starting the oscillation, this circuit doesn't generate oscillation due to the repression of attenuator-TetR aptamer by lacI. First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because TetR aptamer activates Ptet, positive feedback occurs and more and more TetR aptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, TetR aptamer, at the downstream of Attenuator region, is repressed. Then, because new TetR aptamer is not created, the amount of TetR aptamer decreases quickly. Therefore, Ptet is repressed by TetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allows transcription of the downstream (Novick et al, 1989)[6]. The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists vary functions of RNA only by means of nucleotide substitution etc. (Takahashi et al, 2013)[2].<br />
In this paper, many variants of pT181 attenuator/antisense are constructed and the attenuation rate of each variant is different. We chose this mechanism for gene repression. 2013IGKUprojectRNArepressionMECHANISM.png<br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay .<br />
[[File:No-binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
[[File:SPINACHの説明.png]]<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we constructed functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Non-promoter: Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP(GFP generator).<br><br />
[[File:ElectrophoresisRT1.png]]<br />
[[File:ElectrophoresisRT2.png]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-TetRaptamer and antisence-Spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between TetR and TetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay TatR aptamer<br><br />
To confirm the act of TetR aptamer inducing Ptet ,we allow IPTG-inducble TetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, Spinach, no-RNA and attenuator-TetR aptamer. As positive controls, we also use GFP.<br><br />
3, qualitatively Spinach assay (visual recognition & fluorescence microscopes)<br><br />
We check that DFHBI fluorescence on a plate with Spinach.<br><br />
We cultivate IPTG-inducible Spinach in a liquid culture under a shading condition, and add DFHBI. Then we check whether this sample fluorescence after centrifugation. We also check Spinach-GFP and antisense-Spinach.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br><br />
[6][http://www.ncbi.nlm.nih.gov/pubmed/2478296 Novick RP et al. (1999) "pT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator"]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T03:34:32Z<p>Niina: /* RT-PCR */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what makes this difference between dry work and wet work, and makes modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment, the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero. Consequently, this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is adoption of robust gene-circuit model in order to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: Before starting the oscillation, this circuit doesn't generate oscillation due to the repression of attenuator-TetR aptamer by lacI. First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because TetR aptamer activates Ptet, positive feedback occurs and more and more TetR aptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. Therefore, Ptet is repressed by TetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants of pT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay .<br />
[[File:No-binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
[[File:SPINACHの説明.png]]<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we constructed functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Non-promoter: Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of TetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br><br />
[[File:ElectrophoresisRT1.png]]<br />
[[File:ElectrophoresisRT2.png]]<br />
[[File:ElectrophoresisRT3.png]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of TetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-TetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between TetR and TetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay TatR aptamer<br><br />
To confirm the act of TetR aptamer inducing Ptet ,we allow IPTG-inducble TetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-TetR aptamer. As positive controls, we also use GFP.<br><br />
3, qualitatively spinach assay (visual recognition & fluorescence microscopes)<br><br />
We check that DFHBI fluorescence on a plate with spinach.<br><br />
We cultivate IPTG-inducible Spinach in a liquid culture under a shading condition, and add DFHBI. Then we check whether this sample fluorescence after centrifugation. We also check spinach-GFP and antisense-spinach.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/AchievementTeam:Kyoto/Achievement2013-09-28T03:23:34Z<p>Niina: /* safety */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<div class="texts"><br />
=Achievement=<br />
==safety==<br />
We have considered and summarized on safety such as the chassis organisms, their risk groups and the derivation of genes below.<br><br><br />
==ethics==<br />
今までのほとんどのパーツは生物由来のものであったため、遺伝子断片を取り出すために由来となる細胞を殺す必要があったが、今回のパーツは人工的に合成したもので、生物由来でないため由来となる細胞が死ぬことはなく、その点の倫理問題は解消される。問題となってくるのは、生体分子と同じ要素によって構成された人工物を細胞内に組み込むという、人工生命に対する扱いであるが、これは合成生物学が今後も長く付き合っていく問題であろう。<br />
Many genetic parts which have been used are got from living things,so in order to get genetic parts, we must kill cells which have genetic parts we want.But genetic parts we used this time were artificially made,so we did not kill cells to get genetic parts.In our way of experiment,ethical problem that living things are killed to get genetic parts dose not happen.Next problem is that in cells we insert artificial things which have same elements as living things have.As people who study synthetic biology, We must continue to consider about this problem.<br />
<br />
英訳案2<br />
Almost all the parts are from creatures so far, as a result, we have to kill the cells when taking the sections of genes out. Because the parts we used are artificially synthesized this time, there’s no need to kill the cells, so that this ethical problem is avoided. The problem is how we should treat the behavior that integrating artifact of the same composition with biomolecules into cells. This is also a problem synthetic biology will face during a long term.<br />
<br />
In our Human Practice project, we carried out an attitude survey on bioethics which is closely conected with iGEM. And then, a professor, who belongs to Graduate School of Letters, Kyoto University. This leaded to revising subsequent approaches of Human Practice project.<br><br><br />
<br />
==ownership/sharing==<br />
今年のパーツは全て由来が人工合成で、どれも人工合成可能な長さなので、シーケンス情報を共有するだけで全く同じ条件のパーツが作成可能である。人工合成不可能でプラスミドを増やしクローニングを行ってそのものを送らないと共有できない多くのタンパク質のパーツと比べて、shareがより容易になるだろう。<br><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/js}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/AchievementTeam:Kyoto/Achievement2013-09-28T03:23:04Z<p>Niina: </p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<div class="texts"><br />
=Achievement=<br />
==safety==<br />
We have considered and summarized on safety such as the chassis organisms, their risk groups and the derivation of genes below.<br><br><br />
ethics<br><br />
今までのほとんどのパーツは生物由来のものであったため、遺伝子断片を取り出すために由来となる細胞を殺す必要があったが、今回のパーツは人工的に合成したもので、生物由来でないため由来となる細胞が死ぬことはなく、その点の倫理問題は解消される。問題となってくるのは、生体分子と同じ要素によって構成された人工物を細胞内に組み込むという、人工生命に対する扱いであるが、これは合成生物学が今後も長く付き合っていく問題であろう。<br />
Many genetic parts which have been used are got from living things,so in order to get genetic parts, we must kill cells which have genetic parts we want.But genetic parts we used this time were artificially made,so we did not kill cells to get genetic parts.In our way of experiment,ethical problem that living things are killed to get genetic parts dose not happen.Next problem is that in cells we insert artificial things which have same elements as living things have.As people who study synthetic biology, We must continue to consider about this problem.<br />
<br />
英訳案2<br />
Almost all the parts are from creatures so far, as a result, we have to kill the cells when taking the sections of genes out. Because the parts we used are artificially synthesized this time, there’s no need to kill the cells, so that this ethical problem is avoided. The problem is how we should treat the behavior that integrating artifact of the same composition with biomolecules into cells. This is also a problem synthetic biology will face during a long term.<br />
<br />
In our Human Practice project, we carried out an attitude survey on bioethics which is closely conected with iGEM. And then, a professor, who belongs to Graduate School of Letters, Kyoto University. This leaded to revising subsequent approaches of Human Practice project.<br><br><br />
==ownership/sharing==<br />
今年のパーツは全て由来が人工合成で、どれも人工合成可能な長さなので、シーケンス情報を共有するだけで全く同じ条件のパーツが作成可能である。人工合成不可能でプラスミドを増やしクローニングを行ってそのものを送らないと共有できない多くのタンパク質のパーツと比べて、shareがより容易になるだろう。<br><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/js}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T03:21:27Z<p>Niina: /* Future work */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what makes this difference between dry work and wet work, and makes modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants of pT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay .<br />
[[File:No-binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
[[File:SPINACHの説明.png]]<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Non-promoter: Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
3, qualitatively spinach assay (visual recognition & fluorescence microscopes)<br><br />
We check that DFHBI fluorescence on a plate with spinach.<br><br />
We cultivate IPTG-inducible Spinach in a liquid culture under a shading condition, and add DFHBI. Then we check whether this sample fluorescence after centrifugation. We also check spinach-GFP and antisense-spinach.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T03:02:33Z<p>Niina: /* Experiment */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants of pT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay .<br />
[[File:Binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
[[File:SPINACHの説明.png]]<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Non-promoter: Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:59:33Z<p>Niina: /* Reporter */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants of pT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay .<br />
[[File:Binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
[[File:SPINACHの説明.png]]<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:55:59Z<p>Niina: /* Activator */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants of pT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay .<br />
[[File:Binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:55:03Z<p>Niina: /* Activator */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with RNA-RNA interaction as repression mechanism and RNA aptamer-TetR protein interaction as activation mechanism. Fluctuation of factors that effects on a model such as cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay .<br />
[[]][[File:Binding-of-tetR-aptamer.png]]<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:50:56Z<p>Niina: /* Repressor */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]<br />
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>Intending to construct our oscillation circuit, we have to combine two modules into one strand. When we combine two modules, the function of the modules may be inhibited by interactions of secondary structures. In case of RNA it is relatively easier to predict the morecules structure.<br />
We estimated the RNA structure to check whether or not unindicatd duplex formed by open tool using computer.<br />
</p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:47:25Z<p>Niina: /* Oscillation */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyoto_RNA_Prezi.png]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:46:25Z<p>Niina: /* Oscillation */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
[[File:Kyotoscilation1]]<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein. <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:41:50Z<p>Niina: /* Future work */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which represses transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level which vary intensely, which can’t be confirmed via stable protein because RNA is degraded faster than . <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
1. qualitatively assay tatR aptamer<br><br />
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:34:20Z<p>Niina: /* Fusion */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level which vary intensely, which can’t be confirmed via stable protein because RNA is degraded faster than . <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:24:48Z<p>Niina: /* Activator */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level which vary intensely, which can’t be confirmed via stable protein because RNA is degraded faster than . <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:19:34Z<p>Niina: /* Experiment */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
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</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level which vary intensely, which can’t be confirmed via stable protein because RNA is degraded faster than . <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:19:01Z<p>Niina: /* Oscillation */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level which vary intensely, which can’t be confirmed via stable protein because RNA is degraded faster than . <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br />
*Pcon-tetR aptamer-DT<br />
*Pcon-antisense-spinach-DT<br />
*Pcon-spinach-DT<br />
*Pcon-RBS-GFP-DT<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:18:12Z<p>Niina: /* Oscillation */</p>
<hr />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
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</html><br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level which vary intensely, which can’t be confirmed via stable protein because RNA is degraded faster than . <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br />
*Pcon-tetR aptamer-DT<br />
*Pcon-antisense-spinach-DT<br />
*Pcon-spinach-DT<br />
*Pcon-RBS-GFP-DT<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:16:53Z<p>Niina: /* Oscillation */</p>
<hr />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism. Fluctuation of factors that effects on model like cell division can be approximated into zero because the fluctuation become narrower with RNA that is produced or discomposed speedy, we think. We choose spinach as reporter.<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
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</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinach falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level which vary intensely, which can’t be confirmed via stable protein because RNA is degraded faster than . <br />
<br><br><br />
We strongly suggest Spinach aptamer as a reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
[[File:唯一のexperiment.png]]<br />
*Pcon-tetR aptamer-DT<br />
*Pcon-antisense-spinach-DT<br />
*Pcon-spinach-DT<br />
*Pcon-RBS-GFP-DT<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T02:08:29Z<p>Niina: /* Oscillation */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
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<div id="projectRNA"><br />
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<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。というのも、分解や生産の速度がタンパク質よりも速いRNAならば、周期の形成について考えなければならない時間幅が狭くなり、細胞分裂や濃度変動のようなモデルに影響を与える分の変動幅を無視できると考えるからだ。アウトプット機構としては、Spinachを想定する。<br />
We propose following circuit with ncRNA-mRNA interaction as repression mechanism and RNA aptamer-tetR protein interaction as activation mechanism<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDFHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter RNA aptamer,which emits the green fluorescence like GFP when it binds to a fluorophore. It is designed from aptamer combining with the complex--DFHBI, which is a derivative fluorophore of GFP. This fluorophore doesn't work alone That is to say, if fluorescence is conformed after DFHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DFHBI to emit fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which is RNA-based reporter of RNA.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
*Pcon-tetR aptamer-DT<br />
*Pcon-antisense-spinach-DT<br />
*Pcon-spinach-DT<br />
*Pcon-RBS-GFP-DT<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab. Our plans for the construction and assay are shown in [https://2013.igem.org/Kyoto:projectRNA/futureview this page]<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/import|pagename={{PAGENAME}}}}<br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T01:59:28Z<p>Niina: /* Motivation */</p>
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<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. <br />
Then we focused on intracellular condition, and considered what make this difference between dry work and wet work and make modeling and experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment the effect of cell division which seems to give biggest interference with oscillation cycle can be approximated into zero, therefore this circuit is robust enough. From this example, one of the solution to deal with difficulties in reconstructing dry model in wet lab is that constructing robust enough gene-circuit model to ignore the complexity by approximation. However, there are difficulties in choosing factors and under the limitation of remaining the robustness of the cycle. We worked on a consisting oscillation circuit which can be closely reproduced by computer simulation. Our goal is forming oscillation cycle in both wet and dry lab.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
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This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is a derivative fluorophore of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
*Pcon-tetR aptamer-DT<br />
*Pcon-antisense-spinach-DT<br />
*Pcon-spinach-DT<br />
*Pcon-RBS-GFP-DT<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab.<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/ProjectTuringTeam:Kyoto/ProjectTuring2013-09-28T01:46:56Z<p>Niina: /* Result */</p>
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<div class="texts" style="margin-top: -9px;"><br />
=Turing Model<br>-the problems between wet and dry-=<br />
==Introduction==<br />
On Earth, there are various animals which have various patterns on their skin. The mechanism of this pattern formation has not been explained by any valid theories yet, although many hypothesis has been proposed. Among these hypothesis, there is a model pattern called Turing pattern proposed by A. Turing, a famous mathematician *1. S. Kondo *2 and some other researchers *3 suggest that some creatures’ pattern can be explained by Turing’s model. Here we will step by step explain how this Turing pattern is expressed by his model.<br><br />
[[File:IGKU0002.png|300px]]<br><br />
Let’s take a look on a simple hypothetical pattern formed by just two colors. Creatures’ epidermal pattern is expressed on the cells. Let’s assume that the pattern is formed by cells in different state α and β for example. A cell in state α expresses color 1 and changes close cell in state β into state α. Another Cell in state β expresses color 2 and changes close cells in state α into state β, and remote cells in state β into state α. For convenience, hereafter we call the cell in state α {α}, and cell in the state β {β}.<br><br />
[[File:IGKU0003.png|200px]][[File:IGKU0004.png|400px]]<br><br />
Now, we will take a look at the system where two cells {α} and {β} exist uniformly and both of the cells are in the equilibrium state of interaction. Now suppose that the density of {α} and {β} fluctuated somewhere in the system. Assume that the density of cell {β} increases as shown in the center of figure 1. First, {β} in the center changes the neighboring {α} into {β}. And next the same {β} changes the remote {β} into {α}. Then remote {α} changes neighboring {β} into {α}. The pattern forms as this interaction continues<br><br />
[[File:stripeform.gif]]<br><br />
Like this model, a striped pattern is formed by close-and-remote interactions between two states of cells. When we look at this close-and-remote interaction separately, close interaction can be explained as positive feedback reaction in the aspects of polarization. Conversely, the remote interaction can be explained by negative feedback reaction<br><br />
[[File:IGKU0006.png|230px]][[File:IGKU0001.png|300px]]<br><br />
Diffusing substances such as proteins secreted from the cells determine the characters of the cells. It seems that the characters which changes close or remote cells (i.e. &alpha; and &beta;) are function of these diffusing substances. In other words, it can be said that {&alpha;} and {&beda;} secretes different diffusing substances, and the substances lead to close interaction (positive feedback) and remote interaction (negative feedback). Therefore, the pattern formation can be said to be formed by interaction between diffusible substances, as well as cell-cell interaction.<br><br />
[[File:IGKU0008.png|200px]]<br><br />
Then let’s consider about this interactions between diffusible substances in simplified model. Living organism’s body surface consists of cells shaped and sized ununiformly, therefore it is easier to understand if you assume that the body surface is a plane and consists of square cell-units sized uniformly. In this model, we can set diffusible substances which are secreted by {&alpha;} and {&beta;}, and they increase and decrease under the influence of interactions. And then, they are substances which has the same characteristic of cell {&alpha;} and {&beta;} (substances lead to close interaction (positive feedback) and remote interaction (negative feedback)), as a causative agent of the pattern formation on this model surfaces.<br><br />
[[File:IGKU0009.png|150px]][[File:IGKU0011.png|300px]]<br><br />
Then let’s have a look on the interaction between two diffusible substances; one lead to close interaction (positive feedback) and other lead to remote interaction (negative feedback). Hereafter we name this diffusible substances A and B. A has large diffusion velocity and represses B’s increase. B has the small diffusion velocity and promotes both A and B’s increase. If A and B has this characteristic, close interaction (positive feedback) and remote interaction (negative feedback) are formed. A and B forms legato density gradient due to this interaction. When each cell units have the character “Color the appropriate color answering to the denser one among the two diffusible substances inside the cell unit,” the substances’ density gradient can be imagined as pattern of cells.<br />
<br><br />
[[File:IGKU0010.png|500px]]<br><br />
Now let’s consider about how these two diffusible substances interacting each other in each cell unit. The amount of two diffusible substances in each cell unit changes only by diffusion and interaction. Then let’s focus on a certain cell unit (i) and consider about the concentration change. The substances amount change by diffusion is the difference between outflow and inflow. Change by the interaction is dependent on the amount of A and B at the certain moment. <br />
<br><br />
[[File:Hannou.png|400px]]<br><br />
[[File:Hannou2.png|300px]]<br />
<br><br />
Actually, this formula is the same as reaction-diffusion which is proposed by Turing for the purpose of explaining each factors of Turing pattern formation.<br />
It seems to be difficult to understand the content of these formulae. We’re going to explain the content.<br />
<br><br />
Ki, Ki’, and Ki’’ are the constant numbers which indicates how big the influence on interaction of each diffusible substances per unit quantity is. In other words, these terms returns the amount of A and B at a moment depending on the interaction from the amount of A and B at anterior moment. DiA and DiB are the constant numbers peculiar to each diffusible substances which indicates the tendency of diffusion of A and B here. In other words, the terms DiA and DiB is the superficial inflow-outflow budget depending on diffusion of A and B. This is the contents which is described by the equitation.<br />
<br><br />
[[File:IGKU0012.png|300px]][[File:IGKU0013.png|200px]]<br><br />
[[File:IGKU0014.png|300px]][[File:IGKU0015.png|300px]]<br><br />
<br><br />
<br />
==Experiments==<br />
We focused on the constants "Ki, Ki’, Ki’’" in these formulae. These are taken as "always fixed in any point" to Turing pattern. However, in fact, is it true that Ki is always fixed in any point with Turing pattern formed by ''E. coli''? We thought it is not always true in wet work because ''E. coli'' makes A and B. In other words, increase or decrease speed of the amounts of A and B in a certain point depends on the ''E. coli'' density in the point.<br />
<br />
As long as ''E. coli'' is growing ununiformly until a steady state, ''E. coli'' density should be different between each point. This ''E. coli'' density difference makes changes of "Ki, Ki’, Ki’’" between each point.<br><br />
<br />
Can we ignore the "Ki, Ki’, Ki’’" differences? To confirm this, we established these assays.<br><br><br />
1. Confirm expression amount of GFP in both a steady state and a non-steady state with plated ''E. coli'' by common method.<br><br />
[[File:IGKU0020.png|700px]]<br><br />
2. Confirm expression amount of GFP in ''E. coli'' that is activated other protein by IPTG and not activated ''E. coli'' as negative control and check whether expression amount of GFP depends on copy number<br><br />
[[File:IGKU0021.png|500px]]<br><br />
<br />
==Result==<br />
[[File:GuchaFP1.png]]<br><br />
[[File:GuchaFP2.png]]<br><br />
We plated transformant ''E. coli'' containing GFP generator on 2 plates at same time.<br><br />
It is evident that ''E. coli'' density is ununiformly. Moreover, the two plates shows completely different distribution.<br />
<br />
==Discussion==<br />
Thus, when you plate ''E. coli'' by usual method, the ''E. coli'' express GFP ununiformly. This is because you can not plate ''E. coli'' enough uniformly. As long as the expression of GFP is so ununiform, even if you set maximum area of cell-unit which is necessary to generate pattern on a plate, the gaps of average mass of GFP expression between cell-units are large enough. When you set an enough small area to generate pattern, you should plate so uniformly that you can consider the gap of mass of GFP expression between cell-units are small enough. So it is necessary to refine the plating method because the fact that the plates show different distribution means that distribution depends on method of plating. For that, wet lab should plate many times, dry lab should analyze the results every time, reach the minimum area of cell-unit which we can consider the gap of average mass of GFP expression small enough, and provide the dates for we lab. And wet lab refine the method. Thus, if wet lab and dry lab understand enough and go some way along each other, you can construct more accurate and more reliable method.<br><br />
As we have seen, there are factor means ''E. coli'' density we must consider when we think intercellular system. On the other hand, when we think a system inside the cell, the factor ''E. coli'' density is unrelated and do not have to consider. Therefore, after this, we think system inside the cell.<br />
<br />
==Conclusion==<br />
As we showed the example ’Turing Pattern’, the results of wet lab and dry lab are often different because of their lack of understanding and appreciation each other. If both of them provide more information and closely discuss together, wet lab may be able to make an experimental system which imitates the system dry lab approximated to the real system. And wet lab provide quantified datas of a value which are necessary to formularize. If dry lab get these dates, they can create formulae which are well adapted to real system and run well simplified simulation. And if wet lab receive the anticipation date, they will be able to find more interesting results. When dry lab and wet lab join hands like this example ’Turing Pattern’, you can overthrow the future that some experiments should fail. Then, biology would evolve faster.<br />
<br />
==Reference==<br />
1:[http://www.sciencedirect.com/science/article/pii/S0092824005800084 A.M. Turing (1990) "The chemical basis of morphogenesis" Bulletin of Mathmatical Biology Vol. 52, No. 1/2, pp. 153-197]<br><br />
2:<html><a href="http://www.fbs.osaka-u.ac.jp/labs/skondo/paper/kondo%20IJDB%20review.pdf">S. Kondo et al(2009) "How animals get their skin patterns: fish pigment pattern as a live Turing wave" Int. J. Dev. Biol. 53: 851-856</a><br></html><br />
3:<html><a href="http://www.pnas.org/content/106/21/8429.short">Akiko Nakamasu et al(2009) "Interactions between zebrafish pigment cells responsible for the generation of Turing patterns" PNAS vol. 106 no. 21 8429–8434</a><br></html><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/ProjectTuringTeam:Kyoto/ProjectTuring2013-09-28T01:46:14Z<p>Niina: /* Result */</p>
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<div class="texts" style="margin-top: -9px;"><br />
=Turing Model<br>-the problems between wet and dry-=<br />
==Introduction==<br />
On Earth, there are various animals which have various patterns on their skin. The mechanism of this pattern formation has not been explained by any valid theories yet, although many hypothesis has been proposed. Among these hypothesis, there is a model pattern called Turing pattern proposed by A. Turing, a famous mathematician *1. S. Kondo *2 and some other researchers *3 suggest that some creatures’ pattern can be explained by Turing’s model. Here we will step by step explain how this Turing pattern is expressed by his model.<br><br />
[[File:IGKU0002.png|300px]]<br><br />
Let’s take a look on a simple hypothetical pattern formed by just two colors. Creatures’ epidermal pattern is expressed on the cells. Let’s assume that the pattern is formed by cells in different state α and β for example. A cell in state α expresses color 1 and changes close cell in state β into state α. Another Cell in state β expresses color 2 and changes close cells in state α into state β, and remote cells in state β into state α. For convenience, hereafter we call the cell in state α {α}, and cell in the state β {β}.<br><br />
[[File:IGKU0003.png|200px]][[File:IGKU0004.png|400px]]<br><br />
Now, we will take a look at the system where two cells {α} and {β} exist uniformly and both of the cells are in the equilibrium state of interaction. Now suppose that the density of {α} and {β} fluctuated somewhere in the system. Assume that the density of cell {β} increases as shown in the center of figure 1. First, {β} in the center changes the neighboring {α} into {β}. And next the same {β} changes the remote {β} into {α}. Then remote {α} changes neighboring {β} into {α}. The pattern forms as this interaction continues<br><br />
[[File:stripeform.gif]]<br><br />
Like this model, a striped pattern is formed by close-and-remote interactions between two states of cells. When we look at this close-and-remote interaction separately, close interaction can be explained as positive feedback reaction in the aspects of polarization. Conversely, the remote interaction can be explained by negative feedback reaction<br><br />
[[File:IGKU0006.png|230px]][[File:IGKU0001.png|300px]]<br><br />
Diffusing substances such as proteins secreted from the cells determine the characters of the cells. It seems that the characters which changes close or remote cells (i.e. &alpha; and &beta;) are function of these diffusing substances. In other words, it can be said that {&alpha;} and {&beda;} secretes different diffusing substances, and the substances lead to close interaction (positive feedback) and remote interaction (negative feedback). Therefore, the pattern formation can be said to be formed by interaction between diffusible substances, as well as cell-cell interaction.<br><br />
[[File:IGKU0008.png|200px]]<br><br />
Then let’s consider about this interactions between diffusible substances in simplified model. Living organism’s body surface consists of cells shaped and sized ununiformly, therefore it is easier to understand if you assume that the body surface is a plane and consists of square cell-units sized uniformly. In this model, we can set diffusible substances which are secreted by {&alpha;} and {&beta;}, and they increase and decrease under the influence of interactions. And then, they are substances which has the same characteristic of cell {&alpha;} and {&beta;} (substances lead to close interaction (positive feedback) and remote interaction (negative feedback)), as a causative agent of the pattern formation on this model surfaces.<br><br />
[[File:IGKU0009.png|150px]][[File:IGKU0011.png|300px]]<br><br />
Then let’s have a look on the interaction between two diffusible substances; one lead to close interaction (positive feedback) and other lead to remote interaction (negative feedback). Hereafter we name this diffusible substances A and B. A has large diffusion velocity and represses B’s increase. B has the small diffusion velocity and promotes both A and B’s increase. If A and B has this characteristic, close interaction (positive feedback) and remote interaction (negative feedback) are formed. A and B forms legato density gradient due to this interaction. When each cell units have the character “Color the appropriate color answering to the denser one among the two diffusible substances inside the cell unit,” the substances’ density gradient can be imagined as pattern of cells.<br />
<br><br />
[[File:IGKU0010.png|500px]]<br><br />
Now let’s consider about how these two diffusible substances interacting each other in each cell unit. The amount of two diffusible substances in each cell unit changes only by diffusion and interaction. Then let’s focus on a certain cell unit (i) and consider about the concentration change. The substances amount change by diffusion is the difference between outflow and inflow. Change by the interaction is dependent on the amount of A and B at the certain moment. <br />
<br><br />
[[File:Hannou.png|400px]]<br><br />
[[File:Hannou2.png|300px]]<br />
<br><br />
Actually, this formula is the same as reaction-diffusion which is proposed by Turing for the purpose of explaining each factors of Turing pattern formation.<br />
It seems to be difficult to understand the content of these formulae. We’re going to explain the content.<br />
<br><br />
Ki, Ki’, and Ki’’ are the constant numbers which indicates how big the influence on interaction of each diffusible substances per unit quantity is. In other words, these terms returns the amount of A and B at a moment depending on the interaction from the amount of A and B at anterior moment. DiA and DiB are the constant numbers peculiar to each diffusible substances which indicates the tendency of diffusion of A and B here. In other words, the terms DiA and DiB is the superficial inflow-outflow budget depending on diffusion of A and B. This is the contents which is described by the equitation.<br />
<br><br />
[[File:IGKU0012.png|300px]][[File:IGKU0013.png|200px]]<br><br />
[[File:IGKU0014.png|300px]][[File:IGKU0015.png|300px]]<br><br />
<br><br />
<br />
==Experiments==<br />
We focused on the constants "Ki, Ki’, Ki’’" in these formulae. These are taken as "always fixed in any point" to Turing pattern. However, in fact, is it true that Ki is always fixed in any point with Turing pattern formed by ''E. coli''? We thought it is not always true in wet work because ''E. coli'' makes A and B. In other words, increase or decrease speed of the amounts of A and B in a certain point depends on the ''E. coli'' density in the point.<br />
<br />
As long as ''E. coli'' is growing ununiformly until a steady state, ''E. coli'' density should be different between each point. This ''E. coli'' density difference makes changes of "Ki, Ki’, Ki’’" between each point.<br><br />
<br />
Can we ignore the "Ki, Ki’, Ki’’" differences? To confirm this, we established these assays.<br><br><br />
1. Confirm expression amount of GFP in both a steady state and a non-steady state with plated ''E. coli'' by common method.<br><br />
[[File:IGKU0020.png|700px]]<br><br />
2. Confirm expression amount of GFP in ''E. coli'' that is activated other protein by IPTG and not activated ''E. coli'' as negative control and check whether expression amount of GFP depends on copy number<br><br />
[[File:IGKU0021.png|500px]]<br><br />
<br />
==Result==<br />
We plated transformant ''E. coli'' containing GFP generator on 2 plates at same time.<br><br />
It is evident that ''E. coli'' density is ununiformly. Moreover, the two plates shows completely different distribution.<br />
<br />
==Discussion==<br />
Thus, when you plate ''E. coli'' by usual method, the ''E. coli'' express GFP ununiformly. This is because you can not plate ''E. coli'' enough uniformly. As long as the expression of GFP is so ununiform, even if you set maximum area of cell-unit which is necessary to generate pattern on a plate, the gaps of average mass of GFP expression between cell-units are large enough. When you set an enough small area to generate pattern, you should plate so uniformly that you can consider the gap of mass of GFP expression between cell-units are small enough. So it is necessary to refine the plating method because the fact that the plates show different distribution means that distribution depends on method of plating. For that, wet lab should plate many times, dry lab should analyze the results every time, reach the minimum area of cell-unit which we can consider the gap of average mass of GFP expression small enough, and provide the dates for we lab. And wet lab refine the method. Thus, if wet lab and dry lab understand enough and go some way along each other, you can construct more accurate and more reliable method.<br><br />
As we have seen, there are factor means ''E. coli'' density we must consider when we think intercellular system. On the other hand, when we think a system inside the cell, the factor ''E. coli'' density is unrelated and do not have to consider. Therefore, after this, we think system inside the cell.<br />
<br />
==Conclusion==<br />
As we showed the example ’Turing Pattern’, the results of wet lab and dry lab are often different because of their lack of understanding and appreciation each other. If both of them provide more information and closely discuss together, wet lab may be able to make an experimental system which imitates the system dry lab approximated to the real system. And wet lab provide quantified datas of a value which are necessary to formularize. If dry lab get these dates, they can create formulae which are well adapted to real system and run well simplified simulation. And if wet lab receive the anticipation date, they will be able to find more interesting results. When dry lab and wet lab join hands like this example ’Turing Pattern’, you can overthrow the future that some experiments should fail. Then, biology would evolve faster.<br />
<br />
==Reference==<br />
1:[http://www.sciencedirect.com/science/article/pii/S0092824005800084 A.M. Turing (1990) "The chemical basis of morphogenesis" Bulletin of Mathmatical Biology Vol. 52, No. 1/2, pp. 153-197]<br><br />
2:<html><a href="http://www.fbs.osaka-u.ac.jp/labs/skondo/paper/kondo%20IJDB%20review.pdf">S. Kondo et al(2009) "How animals get their skin patterns: fish pigment pattern as a live Turing wave" Int. J. Dev. Biol. 53: 851-856</a><br></html><br />
3:<html><a href="http://www.pnas.org/content/106/21/8429.short">Akiko Nakamasu et al(2009) "Interactions between zebrafish pigment cells responsible for the generation of Turing patterns" PNAS vol. 106 no. 21 8429–8434</a><br></html><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T01:44:21Z<p>Niina: /* Experiment */</p>
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=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br><br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. Then we focused on intracellular condition, and considered about the method to make this difference between modeling and wet experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment, very robust circuit is constructed; cell division which seems to give biggest interference with oscillation cycle can be approximated into zero.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
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<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
*Pcon-tetR aptamer-DT<br />
*Pcon-antisense-spinach-DT<br />
*Pcon-spinach-DT<br />
*Pcon-RBS-GFP-DT<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab.<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T01:42:45Z<p>Niina: /* Reference */</p>
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br><br />
Simulating cell-cell interaction model is too complicated to compute because there is a need to consider about not only intracellular condition but also more complex conditions such as positional relationship. Then we focused on intracellular condition, and considered about the method to make this difference between modeling and wet experiment closer. A study of synthetic biology shows an oscillation model which is confirmed in both dry and wet lab. Under this experiment, very robust circuit is constructed; cell division which seems to give biggest interference with oscillation cycle can be approximated into zero.<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
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<br />
This circuit generates oscillation in the following way: This circuitBefore starting the oscillation, First, tet promoter(Ptet) is repressed by TetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG induces a transcription of TetR aptamer at the downstream of Plac, Spinach, and pT181 antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinach, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, ''E.coli'' in which the united RNA(figC) was introduced expresses more GFP than ''E.coli'' which didn’t have the tetRaptamer sequence(figB). By comparing ''E.coli'' expressing independent tetRaptamer(figA) and ''E.coli'' expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
*tetR aptamer-DT<br />
*antisense-spinach-DT<br />
*spinach-DT<br />
*and GFP generator<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab.<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:KyotoTeam:Kyoto2013-09-28T01:37:07Z<p>Niina: </p>
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{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/ProjectTuringTeam:Kyoto/ProjectTuring2013-09-28T01:35:05Z<p>Niina: /* Discussion */</p>
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<div class="texts" style="margin-top: -9px;"><br />
=Turing Model<br>-the problems between wet and dry-=<br />
==Introduction==<br />
On Earth, there are various animals which have various patterns on their skin. The mechanism of this pattern formation has not been explained by any valid theories yet, although many hypothesis has been proposed. Among these hypothesis, there is a model pattern called Turing pattern proposed by A. Turing, a famous mathematician *1. S. Kondo *2 and some other researchers *3 suggest that some creatures’ pattern can be explained by Turing’s model. Here we will step by step explain how this Turing pattern is expressed by his model.<br><br />
[[File:IGKU0002.png|300px]]<br><br />
Let’s take a look on a simple hypothetical pattern formed by just two colors. Creatures’ epidermal pattern is expressed on the cells. Let’s assume that the pattern is formed by cells in different state α and β for example. A cell in state α expresses color 1 and changes close cell in state β into state α. Another Cell in state β expresses color 2 and changes close cells in state α into state β, and remote cells in state β into state α. For convenience, hereafter we call the cell in state α {α}, and cell in the state β {β}.<br><br />
[[File:IGKU0003.png|200px]][[File:IGKU0004.png|400px]]<br><br />
Now, we will take a look at the system where two cells {α} and {β} exist uniformly and both of the cells are in the equilibrium state of interaction. Now suppose that the density of {α} and {β} fluctuated somewhere in the system. Assume that the density of cell {β} increases as shown in the center of figure 1. First, {β} in the center changes the neighboring {α} into {β}. And next the same {β} changes the remote {β} into {α}. Then remote {α} changes neighboring {β} into {α}. The pattern forms as this interaction continues<br><br />
[[File:stripeform.gif]]<br><br />
Like this model, a striped pattern is formed by close-and-remote interactions between two states of cells. When we look at this close-and-remote interaction separately, close interaction can be explained as positive feedback reaction in the aspects of polarization. Conversely, the remote interaction can be explained by negative feedback reaction<br><br />
[[File:IGKU0006.png|230px]][[File:IGKU0001.png|300px]]<br><br />
Diffusing substances such as proteins secreted from the cells determine the characters of the cells. It seems that the characters which changes close or remote cells (i.e. &alpha; and &beta;) are function of these diffusing substances. In other words, it can be said that {&alpha;} and {&beda;} secretes different diffusing substances, and the substances lead to close interaction (positive feedback) and remote interaction (negative feedback). Therefore, the pattern formation can be said to be formed by interaction between diffusible substances, as well as cell-cell interaction.<br><br />
[[File:IGKU0008.png|200px]]<br><br />
Then let’s consider about this interactions between diffusible substances in simplified model. Living organism’s body surface consists of cells shaped and sized ununiformly, therefore it is easier to understand if you assume that the body surface is a plane and consists of square cell-units sized uniformly. In this model, we can set diffusible substances which are secreted by {&alpha;} and {&beta;}, and they increase and decrease under the influence of interactions. And then, they are substances which has the same characteristic of cell {&alpha;} and {&beta;} (substances lead to close interaction (positive feedback) and remote interaction (negative feedback)), as a causative agent of the pattern formation on this model surfaces.<br><br />
[[File:IGKU0009.png|150px]][[File:IGKU0011.png|300px]]<br><br />
Then let’s have a look on the interaction between two diffusible substances; one lead to close interaction (positive feedback) and other lead to remote interaction (negative feedback). Hereafter we name this diffusible substances A and B. A has large diffusion velocity and represses B’s increase. B has the small diffusion velocity and promotes both A and B’s increase. If A and B has this characteristic, close interaction (positive feedback) and remote interaction (negative feedback) are formed. A and B forms legato density gradient due to this interaction. When each cell units have the character “Color the appropriate color answering to the denser one among the two diffusible substances inside the cell unit,” the substances’ density gradient can be imagined as pattern of cells.<br />
<br><br />
[[File:IGKU0010.png|500px]]<br><br />
Now let’s consider about how these two diffusible substances interacting each other in each cell unit. The amount of two diffusible substances in each cell unit changes only by diffusion and interaction. Then let’s focus on a certain cell unit (i) and consider about the concentration change. The substances amount change by diffusion is the difference between outflow and inflow. Change by the interaction is dependent on the amount of A and B at the certain moment. <br />
<br><br />
[[File:Hannou.png|400px]]<br><br />
[[File:Hannou2.png|300px]]<br />
<br><br />
Actually, this formula is the same as reaction-diffusion which is proposed by Turing for the purpose of explaining each factors of Turing pattern formation.<br />
It seems to be difficult to understand the content of these formulae. We’re going to explain the content.<br />
<br><br />
Ki, Ki’, and Ki’’ are the constant numbers which indicates how big the influence on interaction of each diffusible substances per unit quantity is. In other words, these terms returns the amount of A and B at a moment depending on the interaction from the amount of A and B at anterior moment. DiA and DiB are the constant numbers peculiar to each diffusible substances which indicates the tendency of diffusion of A and B here. In other words, the terms DiA and DiB is the superficial inflow-outflow budget depending on diffusion of A and B. This is the contents which is described by the equitation.<br />
<br><br />
[[File:IGKU0012.png|300px]][[File:IGKU0013.png|200px]]<br><br />
[[File:IGKU0014.png|300px]][[File:IGKU0015.png|300px]]<br><br />
<br><br />
<br />
==Experiments==<br />
We focused on the constants "Ki, Ki’, Ki’’" in these formulae. These are taken as "always fixed in any point" to Turing pattern. However, in fact, is it true that Ki is always fixed in any point with Turing pattern formed by ''E. coli''? We thought it is not always true in wet work because ''E. coli'' makes A and B. In other words, increase or decrease speed of the amounts of A and B in a certain point depends on the ''E. coli'' density in the point.<br />
<br />
As long as ''E. coli'' is growing not uniformly until a steady state, ''E. coli'' density should be different between each point. This ''E. coli'' density difference makes "Ki, Ki’, Ki’’" change between each point.<br><br />
<br />
Can we ignore the "Ki, Ki’, Ki’’" differences? To confirm this, we established these assays.<br><br><br />
1. Confirm expression amount of GFP in both a steady state and a non-steady state with plated ''E. coli'' by common method.<br><br />
[[File:IGKU0020.png|700px]]<br><br />
2. Confirm expression amount of GFP in ''E. coli'' that is activated other protein by IPTG and not activated ''E. coli'' as negative control and check whether expression amount of GFP depends on copy number<br><br />
[[File:IGKU0021.png|500px]]<br><br />
<br />
==Result==<br />
[[File:GuchaguchaFluorescenceProtein1.png]]<br><br />
[[File:GuchaguchaFluorescenceProtein2.png]]<br><br />
We plated transformant ''E. coli'' containing GFP generator on 2 plates at same time.<br><br />
It is evident that ''E. coli'' density is ununiformly. Moreover, the two plates shows completely different distribution.<br />
<br />
==Discussion==<br />
Thus, when you plate ''E. coli'' by usual method, the ''E. coli'' express GFP ununiformly. This is because you can not plate ''E. coli'' enough uniformly. As long as the expression of GFP is so ununiform, even if you set maximum area of cell-unit which is necessary to generate pattern on a plate, the gaps of average mass of GFP expression between cell-units are large enough. When you set an enough small area to generate pattern, you should plate so uniformly that you can consider the gap of mass of GFP expression between cell-units are small enough. So it is necessary to refine the plating method because the fact that the plates show different distribution means that distribution depends on method of plating. For that, wet lab should plate many times, dry lab should analyze the results every time, reach the minimum area of cell-unit which we can consider the gap of average mass of GFP expression small enough, and provide the dates for we lab. And wet lab refine the method. Thus, if wet lab and dry lab understand enough and go some way along each other, you can construct more accurate and more reliable method.<br><br />
As we have seen, there are factor means ''E. coli'' density we must consider when we think intercellular system. On the other hand, when we think a system inside the cell, the factor ''E. coli'' density is unrelated and do not have to consider. Therefore, after this, we think system inside the cell.<br />
<br />
==Conclusion==<br />
As we showed the example ’Turing Pattern’, the results of wet lab and dry lab are often different because of their lack of understanding and appreciation each other. If both of them provide more information and closely discuss together, wet lab may be able to make an experimental system which imitates the system dry lab approximated to the real system. And wet lab provide quantified datas of a value which are necessary to formularize. If dry lab get these dates, they can create formulae which are well adapted to real system and run well simplified simulation. And if wet lab receive the anticipation date, they will be able to find more interesting results. When dry lab and wet lab join hands like this example ’Turing Pattern’, you can overthrow the future that some experiments should fail. Then, biology would evolve faster.<br />
<br />
==Reference==<br />
1:[http://www.sciencedirect.com/science/article/pii/S0092824005800084 A.M. Turing (1990) "The chemical basis of morphogenesis" Bulletin of Mathmatical Biology Vol. 52, No. 1/2, pp. 153-197]<br><br />
2:<html><a href="http://www.fbs.osaka-u.ac.jp/labs/skondo/paper/kondo%20IJDB%20review.pdf">S. Kondo et al(2009) "How animals get their skin patterns: fish pigment pattern as a live Turing wave" Int. J. Dev. Biol. 53: 851-856</a><br></html><br />
3:<html><a href="http://www.pnas.org/content/106/21/8429.short">Akiko Nakamasu et al(2009) "Interactions between zebrafish pigment cells responsible for the generation of Turing patterns" PNAS vol. 106 no. 21 8429–8434</a><br></html><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/ProjectTuringTeam:Kyoto/ProjectTuring2013-09-28T01:22:42Z<p>Niina: /* Result */</p>
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</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=Turing Model<br>-the problems between wet and dry-=<br />
==Introduction==<br />
On Earth, there are various animals which have various patterns on their skin. The mechanism of this pattern formation has not been explained by any valid theories yet, although many hypothesis has been proposed. Among these hypothesis, there is a model pattern called Turing pattern proposed by A. Turing, a famous mathematician *1. S. Kondo *2 and some other researchers *3 suggest that some creatures’ pattern can be explained by Turing’s model. Here we will step by step explain how this Turing pattern is expressed by his model.<br><br />
[[File:IGKU0002.png|300px]]<br><br />
Let’s take a look on a simple hypothetical pattern formed by just two colors. Creatures’ epidermal pattern is expressed on the cells. Let’s assume that the pattern is formed by cells in different state α and β for example. A cell in state α expresses color 1 and changes close cell in state β into state α. Another Cell in state β expresses color 2 and changes close cells in state α into state β, and remote cells in state β into state α. For convenience, hereafter we call the cell in state α {α}, and cell in the state β {β}.<br><br />
[[File:IGKU0003.png|200px]][[File:IGKU0004.png|400px]]<br><br />
Now, we will take a look at the system where two cells {α} and {β} exist uniformly and both of the cells are in the equilibrium state of interaction. Now suppose that the density of {α} and {β} fluctuated somewhere in the system. Assume that the density of cell {β} increases as shown in the center of figure 1. First, {β} in the center changes the neighboring {α} into {β}. And next the same {β} changes the remote {β} into {α}. Then remote {α} changes neighboring {β} into {α}. The pattern forms as this interaction continues<br><br />
[[File:stripeform.gif]]<br><br />
Like this model, a striped pattern is formed by close-and-remote interactions between two states of cells. When we look at this close-and-remote interaction separately, close interaction can be explained as positive feedback reaction in the aspects of polarization. Conversely, the remote interaction can be explained by negative feedback reaction<br><br />
[[File:IGKU0006.png|230px]][[File:IGKU0001.png|300px]]<br><br />
Diffusing substances such as proteins secreted from the cells determine the characters of the cells. It seems that the characters which changes close or remote cells (i.e. &alpha; and &beta;) are function of these diffusing substances. In other words, it can be said that {&alpha;} and {&beda;} secretes different diffusing substances, and the substances lead to close interaction (positive feedback) and remote interaction (negative feedback). Therefore, the pattern formation can be said to be formed by interaction between diffusible substances, as well as cell-cell interaction.<br><br />
[[File:IGKU0008.png|200px]]<br><br />
Then let’s consider about this interactions between diffusible substances in simplified model. Living organism’s body surface consists of cells shaped and sized ununiformly, therefore it is easier to understand if you assume that the body surface is a plane and consists of square cell-units sized uniformly. In this model, we can set diffusible substances which are secreted by {&alpha;} and {&beta;}, and they increase and decrease under the influence of interactions. And then, they are substances which has the same characteristic of cell {&alpha;} and {&beta;} (substances lead to close interaction (positive feedback) and remote interaction (negative feedback)), as a causative agent of the pattern formation on this model surfaces.<br><br />
[[File:IGKU0009.png|150px]][[File:IGKU0011.png|300px]]<br><br />
Then let’s have a look on the interaction between two diffusible substances; one lead to close interaction (positive feedback) and other lead to remote interaction (negative feedback). Hereafter we name this diffusible substances A and B. A has large diffusion velocity and represses B’s increase. B has the small diffusion velocity and promotes both A and B’s increase. If A and B has this characteristic, close interaction (positive feedback) and remote interaction (negative feedback) are formed. A and B forms legato density gradient due to this interaction. When each cell units have the character “Color the appropriate color answering to the denser one among the two diffusible substances inside the cell unit,” the substances’ density gradient can be imagined as pattern of cells.<br />
<br><br />
[[File:IGKU0010.png|500px]]<br><br />
Now let’s consider about how these two diffusible substances interacting each other in each cell unit. The amount of two diffusible substances in each cell unit changes only by diffusion and interaction. Then let’s focus on a certain cell unit (i) and consider about the concentration change. The substances amount change by diffusion is the difference between outflow and inflow. Change by the interaction is dependent on the amount of A and B at the certain moment. <br />
<br><br />
[[File:Hannou.png|400px]]<br><br />
[[File:Hannou2.png|300px]]<br />
<br><br />
Actually, this formula is the same as reaction-diffusion which is proposed by Turing for the purpose of explaining each factors of Turing pattern formation.<br />
It seems to be difficult to understand the content of these formulae. We’re going to explain the content.<br />
<br><br />
Ki, Ki’, and Ki’’ are the constant numbers which indicates how big the influence on interaction of each diffusible substances per unit quantity is. In other words, these terms returns the amount of A and B at a moment depending on the interaction from the amount of A and B at anterior moment. DiA and DiB are the constant numbers peculiar to each diffusible substances which indicates the tendency of diffusion of A and B here. In other words, the terms DiA and DiB is the superficial inflow-outflow budget depending on diffusion of A and B. This is the contents which is described by the equitation.<br />
<br><br />
[[File:IGKU0012.png|300px]][[File:IGKU0013.png|200px]]<br><br />
[[File:IGKU0014.png|300px]][[File:IGKU0015.png|300px]]<br><br />
<br><br />
<br />
==Experiments==<br />
We focused on the constants "Ki, Ki’, Ki’’" in these formulae. These are taken as "always fixed in any point" to Turing pattern. However, in fact, is it true that Ki is always fixed in any point with Turing pattern formed by E. coli? We thought it is not always true in wet work because E. coli makes A and B. In other words, increase or decrease speed of the amounts of A and B in a certain point depends on the E. coli density in the point.<br />
<br />
As long as E. coli is growing not uniformly until a steady state, E. coli density should be different between each point. This ''E.coli'' density difference makes "Ki, Ki’, Ki’’" change between each point.<br><br />
<br />
Can we ignore the "Ki, Ki’, Ki’’" differences? To confirm this, we established these assays.<br><br><br />
1. Confirm expression amount of GFP in both a steady state and a non-steady state with plated E. coli by common method.<br><br />
[[File:IGKU0020.png|700px]]<br><br />
2. Confirm expression amount of GFP in E. coli that is activated other protein by IPTG and not activated ''E.coli'' as negative control and check whether expression amount of GFP depends on copy number<br><br />
[[File:IGKU0021.png|500px]]<br><br />
<br />
==Result==<br />
[[File:GuchaguchaFluorescenceProtein1.png]]<br><br />
[[File:GuchaguchaFluorescenceProtein2.png]]<br><br />
We plated transformant E.coli containing GFP generator on 2 plates at same time.<br><br />
It is evident that E. coli density is ununiformly. Moreover, the two plates shows completely different distribution.<br />
<br />
==Discussion==<br />
Thus, when you plate E. coli by usual method, the E. coli express GFP ununiformly. This is because you can not plate E. coli enough uniformly. As long as the expression of GFP is so ununiform, even if you set maximum area of cell-unit which is necessary to generate pattern on a plate, the gaps of average mass of GFP expression between cell-units are large enough. When you set an enough small area to generate pattern, you should plate so uniformly that you can consider the gap of mass of GFP expression between cell-units are small enough. So it is necessary to refine the plating method. For that, wet lab should plate many times, dry lab should analyze the results every time, reach the minimum area of cell-unit which we can consider the gap of average mass of GFP expression small enough, and provide the dates for we lab. And wet lab refine the method. Thus, if wet lab and dry lab understand enough and go some way along each other, you can construct more accurate and more reliable method.<br><br />
As we have seen, there are factor means E. coli density we must consider when we think intercellular system. On the other hand, when we think a system inside the cell, the factor E. coli density is unrelated and do not have to consider. Therefore, after this, we think system inside the cell.<br />
<br />
==Conclusion==<br />
As we showed the example ’Turing Pattern’, the results of wet lab and dry lab are often different because of their lack of understanding and appreciation each other. If both of them provide more information and closely discuss together, wet lab may be able to make an experimental system which imitates the system dry lab approximated to the real system. And wet lab provide quantified datas of a value which are necessary to formularize. If dry lab get these dates, they can create formulae which are well adapted to real system and run well simplified simulation. And if wet lab receive the anticipation date, they will be able to find more interesting results. When dry lab and wet lab join hands like this example ’Turing Pattern’, you can overthrow the future that some experiments should fail. Then, biology would evolve faster.<br />
<br />
==Reference==<br />
1:[http://www.sciencedirect.com/science/article/pii/S0092824005800084 A.M. Turing (1990) "The chemical basis of morphogenesis" Bulletin of Mathmatical Biology Vol. 52, No. 1/2, pp. 153-197]<br><br />
2:<html><a href="http://www.fbs.osaka-u.ac.jp/labs/skondo/paper/kondo%20IJDB%20review.pdf">S. Kondo et al(2009) "How animals get their skin patterns: fish pigment pattern as a live Turing wave" Int. J. Dev. Biol. 53: 851-856</a><br></html><br />
3:<html><a href="http://www.pnas.org/content/106/21/8429.short">Akiko Nakamasu et al(2009) "Interactions between zebrafish pigment cells responsible for the generation of Turing patterns" PNAS vol. 106 no. 21 8429–8434</a><br></html><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/ProjectTuringTeam:Kyoto/ProjectTuring2013-09-28T01:16:40Z<p>Niina: /* Result */</p>
<hr />
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<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
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</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=Turing Model<br>-the problems between wet and dry-=<br />
==Introduction==<br />
On Earth, there are various animals which have various patterns on their skin. The mechanism of this pattern formation has not been explained by any valid theories yet, although many hypothesis has been proposed. Among these hypothesis, there is a model pattern called Turing pattern proposed by A. Turing, a famous mathematician *1. S. Kondo *2 and some other researchers *3 suggest that some creatures’ pattern can be explained by Turing’s model. Here we will step by step explain how this Turing pattern is expressed by his model.<br><br />
[[File:IGKU0002.png|300px]]<br><br />
Let’s take a look on a simple hypothetical pattern formed by just two colors. Creatures’ epidermal pattern is expressed on the cells. Let’s assume that the pattern is formed by cells in different state α and β for example. A cell in state α expresses color 1 and changes close cell in state β into state α. Another Cell in state β expresses color 2 and changes close cells in state α into state β, and remote cells in state β into state α. For convenience, hereafter we call the cell in state α {α}, and cell in the state β {β}.<br><br />
[[File:IGKU0003.png|200px]][[File:IGKU0004.png|400px]]<br><br />
Now, we will take a look at the system where two cells {α} and {β} exist uniformly and both of the cells are in the equilibrium state of interaction. Now suppose that the density of {α} and {β} fluctuated somewhere in the system. Assume that the density of cell {β} increases as shown in the center of figure 1. First, {β} in the center changes the neighboring {α} into {β}. And next the same {β} changes the remote {β} into {α}. Then remote {α} changes neighboring {β} into {α}. The pattern forms as this interaction continues<br><br />
[[File:stripeform.gif]]<br><br />
Like this model, a striped pattern is formed by close-and-remote interactions between two states of cells. When we look at this close-and-remote interaction separately, close interaction can be explained as positive feedback reaction in the aspects of polarization. Conversely, the remote interaction can be explained by negative feedback reaction<br><br />
[[File:IGKU0006.png|230px]][[File:IGKU0001.png|300px]]<br><br />
Diffusing substances such as proteins secreted from the cells determine the characters of the cells. It seems that the characters which changes close or remote cells (i.e. &alpha; and &beta;) are function of these diffusing substances. In other words, it can be said that {&alpha;} and {&beda;} secretes different diffusing substances, and the substances lead to close interaction (positive feedback) and remote interaction (negative feedback). Therefore, the pattern formation can be said to be formed by interaction between diffusible substances, as well as cell-cell interaction.<br><br />
[[File:IGKU0008.png|200px]]<br><br />
Then let’s consider about this interactions between diffusible substances in simplified model. Living organism’s body surface consists of cells shaped and sized ununiformly, therefore it is easier to understand if you assume that the body surface is a plane and consists of square cell-units sized uniformly. In this model, we can set diffusible substances which are secreted by {&alpha;} and {&beta;}, and they increase and decrease under the influence of interactions. And then, they are substances which has the same characteristic of cell {&alpha;} and {&beta;} (substances lead to close interaction (positive feedback) and remote interaction (negative feedback)), as a causative agent of the pattern formation on this model surfaces.<br><br />
[[File:IGKU0009.png|150px]][[File:IGKU0011.png|300px]]<br><br />
Then let’s have a look on the interaction between two diffusible substances; one lead to close interaction (positive feedback) and other lead to remote interaction (negative feedback). Hereafter we name this diffusible substances A and B. A has large diffusion velocity and represses B’s increase. B has the small diffusion velocity and promotes both A and B’s increase. If A and B has this characteristic, close interaction (positive feedback) and remote interaction (negative feedback) are formed. A and B forms legato density gradient due to this interaction. When each cell units have the character “Color the appropriate color answering to the denser one among the two diffusible substances inside the cell unit,” the substances’ density gradient can be imagined as pattern of cells.<br />
<br><br />
[[File:IGKU0010.png|500px]]<br><br />
Now let’s consider about how these two diffusible substances interacting each other in each cell unit. The amount of two diffusible substances in each cell unit changes only by diffusion and interaction. Then let’s focus on a certain cell unit (i) and consider about the concentration change. The substances amount change by diffusion is the difference between outflow and inflow. Change by the interaction is dependent on the amount of A and B at the certain moment. <br />
<br><br />
[[File:Hannou.png|400px]]<br><br />
[[File:Hannou2.png|300px]]<br />
<br><br />
Actually, this formula is the same as reaction-diffusion which is proposed by Turing for the purpose of explaining each factors of Turing pattern formation.<br />
It seems to be difficult to understand the content of these formulae. We’re going to explain the content.<br />
<br><br />
Ki, Ki’, and Ki’’ are the constant numbers which indicates how big the influence on interaction of each diffusible substances per unit quantity is. In other words, these terms returns the amount of A and B at a moment depending on the interaction from the amount of A and B at anterior moment. DiA and DiB are the constant numbers peculiar to each diffusible substances which indicates the tendency of diffusion of A and B here. In other words, the terms DiA and DiB is the superficial inflow-outflow budget depending on diffusion of A and B. This is the contents which is described by the equitation.<br />
<br><br />
[[File:IGKU0012.png|300px]][[File:IGKU0013.png|200px]]<br><br />
[[File:IGKU0014.png|300px]][[File:IGKU0015.png|300px]]<br><br />
<br><br />
<br />
==Experiments==<br />
We focused on the constants "Ki, Ki’, Ki’’" in these formulae. These are taken as "always fixed in any point" to Turing pattern. However, in fact, is it true that Ki is always fixed in any point with Turing pattern formed by E. coli? We thought it is not always true in wet work because E. coli makes A and B. In other words, increase or decrease speed of the amounts of A and B in a certain point depends on the E. coli density in the point.<br />
<br />
As long as E. coli is growing not uniformly until a steady state, E. coli density should be different between each point. This ''E.coli'' density difference makes "Ki, Ki’, Ki’’" change between each point.<br><br />
<br />
Can we ignore the "Ki, Ki’, Ki’’" differences? To confirm this, we established these assays.<br><br><br />
1. Confirm expression amount of GFP in both a steady state and a non-steady state with plated E. coli by common method.<br><br />
[[File:IGKU0020.png|700px]]<br><br />
2. Confirm expression amount of GFP in E. coli that is activated other protein by IPTG and not activated ''E.coli'' as negative control and check whether expression amount of GFP depends on copy number<br><br />
[[File:IGKU0021.png|500px]]<br><br />
<br />
==Result==<br />
[[File:GuchaguchaFluorescenceProtein1.png]]<br><br />
[[File:GuchaguchaFluorescenceProtein2.png]]<br><br />
We plated transformant E.coli containing GFP generator on 2 plates at same time.<br><br />
It is evident that<br />
<br />
==Discussion==<br />
Thus, when you plate E. coli by usual method, the E. coli express GFP ununiformly. This is because you can not plate E. coli enough uniformly. As long as the expression of GFP is so ununiform, even if you set maximum area of cell-unit which is necessary to generate pattern on a plate, the gaps of average mass of GFP expression between cell-units are large enough. When you set an enough small area to generate pattern, you should plate so uniformly that you can consider the gap of mass of GFP expression between cell-units are small enough. So it is necessary to refine the plating method. For that, wet lab should plate many times, dry lab should analyze the results every time, reach the minimum area of cell-unit which we can consider the gap of average mass of GFP expression small enough, and provide the dates for we lab. And wet lab refine the method. Thus, if wet lab and dry lab understand enough and go some way along each other, you can construct more accurate and more reliable method.<br><br />
As we have seen, there are factor means E. coli density we must consider when we think intercellular system. On the other hand, when we think a system inside the cell, the factor E. coli density is unrelated and do not have to consider. Therefore, after this, we think system inside the cell.<br />
<br />
==Conclusion==<br />
As we showed the example ’Turing Pattern’, the results of wet lab and dry lab are often different because of their lack of understanding and appreciation each other. If both of them provide more information and closely discuss together, wet lab may be able to make an experimental system which imitates the system dry lab approximated to the real system. And wet lab provide quantified datas of a value which are necessary to formularize. If dry lab get these dates, they can create formulae which are well adapted to real system and run well simplified simulation. And if wet lab receive the anticipation date, they will be able to find more interesting results. When dry lab and wet lab join hands like this example ’Turing Pattern’, you can overthrow the future that some experiments should fail. Then, biology would evolve faster.<br />
<br />
==Reference==<br />
1:[http://www.sciencedirect.com/science/article/pii/S0092824005800084 A.M. Turing (1990) "The chemical basis of morphogenesis" Bulletin of Mathmatical Biology Vol. 52, No. 1/2, pp. 153-197]<br><br />
2:<html><a href="http://www.fbs.osaka-u.ac.jp/labs/skondo/paper/kondo%20IJDB%20review.pdf">S. Kondo et al(2009) "How animals get their skin patterns: fish pigment pattern as a live Turing wave" Int. J. Dev. Biol. 53: 851-856</a><br></html><br />
3:<html><a href="http://www.pnas.org/content/106/21/8429.short">Akiko Nakamasu et al(2009) "Interactions between zebrafish pigment cells responsible for the generation of Turing patterns" PNAS vol. 106 no. 21 8429–8434</a><br></html><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/ProjectTuringTeam:Kyoto/ProjectTuring2013-09-28T01:08:21Z<p>Niina: /* Result */</p>
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<div class="texts" style="margin-top: -9px;"><br />
=Turing Model<br>-the problems between wet and dry-=<br />
==Introduction==<br />
On Earth, there are various animals which have various patterns on their skin. The mechanism of this pattern formation has not been explained by any valid theories yet, although many hypothesis has been proposed. Among these hypothesis, there is a model pattern called Turing pattern proposed by A. Turing, a famous mathematician *1. S. Kondo *2 and some other researchers *3 suggest that some creatures’ pattern can be explained by Turing’s model. Here we will step by step explain how this Turing pattern is expressed by his model.<br><br />
[[File:IGKU0002.png|300px]]<br><br />
Let’s take a look on a simple hypothetical pattern formed by just two colors. Creatures’ epidermal pattern is expressed on the cells. Let’s assume that the pattern is formed by cells in different state α and β for example. A cell in state α expresses color 1 and changes close cell in state β into state α. Another Cell in state β expresses color 2 and changes close cells in state α into state β, and remote cells in state β into state α. For convenience, hereafter we call the cell in state α {α}, and cell in the state β {β}.<br><br />
[[File:IGKU0003.png|200px]][[File:IGKU0004.png|400px]]<br><br />
Now, we will take a look at the system where two cells {α} and {β} exist uniformly and both of the cells are in the equilibrium state of interaction. Now suppose that the density of {α} and {β} fluctuated somewhere in the system. Assume that the density of cell {β} increases as shown in the center of figure 1. First, {β} in the center changes the neighboring {α} into {β}. And next the same {β} changes the remote {β} into {α}. Then remote {α} changes neighboring {β} into {α}. The pattern forms as this interaction continues<br><br />
[[File:stripeform.gif]]<br><br />
Like this model, a striped pattern is formed by close-and-remote interactions between two states of cells. When we look at this close-and-remote interaction separately, close interaction can be explained as positive feedback reaction in the aspects of polarization. Conversely, the remote interaction can be explained by negative feedback reaction<br><br />
[[File:IGKU0006.png|230px]][[File:IGKU0001.png|300px]]<br><br />
Diffusing substances such as proteins secreted from the cells determine the characters of the cells. It seems that the characters which changes close or remote cells (i.e. &alpha; and &beta;) are function of these diffusing substances. In other words, it can be said that {&alpha;} and {&beda;} secretes different diffusing substances, and the substances lead to close interaction (positive feedback) and remote interaction (negative feedback). Therefore, the pattern formation can be said to be formed by interaction between diffusible substances, as well as cell-cell interaction.<br><br />
[[File:IGKU0008.png|200px]]<br><br />
Then let’s consider about this interactions between diffusible substances in simplified model. Living organism’s body surface consists of cells shaped and sized ununiformly, therefore it is easier to understand if you assume that the body surface is a plane and consists of square cell-units sized uniformly. In this model, we can set diffusible substances which are secreted by {&alpha;} and {&beta;}, and they increase and decrease under the influence of interactions. And then, they are substances which has the same characteristic of cell {&alpha;} and {&beta;} (substances lead to close interaction (positive feedback) and remote interaction (negative feedback)), as a causative agent of the pattern formation on this model surfaces.<br><br />
[[File:IGKU0009.png|150px]][[File:IGKU0011.png|300px]]<br><br />
Then let’s have a look on the interaction between two diffusible substances; one lead to close interaction (positive feedback) and other lead to remote interaction (negative feedback). Hereafter we name this diffusible substances A and B. A has large diffusion velocity and represses B’s increase. B has the small diffusion velocity and promotes both A and B’s increase. If A and B has this characteristic, close interaction (positive feedback) and remote interaction (negative feedback) are formed. A and B forms legato density gradient due to this interaction. When each cell units have the character “Color the appropriate color answering to the denser one among the two diffusible substances inside the cell unit,” the substances’ density gradient can be imagined as pattern of cells.<br />
<br><br />
[[File:IGKU0010.png|500px]]<br><br />
Now let’s consider about how these two diffusible substances interacting each other in each cell unit. The amount of two diffusible substances in each cell unit changes only by diffusion and interaction. Then let’s focus on a certain cell unit (i) and consider about the concentration change. The substances amount change by diffusion is the difference between outflow and inflow. Change by the interaction is dependent on the amount of A and B at the certain moment. <br />
<br><br />
[[File:Hannou.png|400px]]<br><br />
[[File:Hannou2.png|300px]]<br />
<br><br />
Actually, this formula is the same as reaction-diffusion which is proposed by Turing for the purpose of explaining each factors of Turing pattern formation.<br />
It seems to be difficult to understand the content of these formulae. We’re going to explain the content.<br />
<br><br />
Ki, Ki’, and Ki’’ are the constant numbers which indicates how big the influence on interaction of each diffusible substances per unit quantity is. In other words, these terms returns the amount of A and B at a moment depending on the interaction from the amount of A and B at anterior moment. DiA and DiB are the constant numbers peculiar to each diffusible substances which indicates the tendency of diffusion of A and B here. In other words, the terms DiA and DiB is the superficial inflow-outflow budget depending on diffusion of A and B. This is the contents which is described by the equitation.<br />
<br><br />
[[File:IGKU0012.png|300px]][[File:IGKU0013.png|200px]]<br><br />
[[File:IGKU0014.png|300px]][[File:IGKU0015.png|300px]]<br><br />
<br><br />
<br />
==Experiments==<br />
We focused on the constants "Ki, Ki’, Ki’’" in these formulae. These are taken as "always fixed in any point" to Turing pattern. However, in fact, it is true that Ki is always fixed in any point with Turing pattern formed by E. coli. We thought it is not always true in wet work because E. coli makes A and B. In other words, increase or decrease speed of the amount of A and B in a certain point depends on the E. coli density in the point.<br />
<br />
As long as E. coli is growing not uniformly until a steady state, E. coli density should be different between each point. This E.coli density difference makes "Ki, Ki’, Ki’’" change between each point.<br><br />
<br />
Can we ignore the "Ki, Ki’, Ki’’" differences? To confirm this, we established these assays.<br><br><br />
<br />
1. Confirm expression amount of GFP in both a steady state and a non-steady state with plated E. coli by common method.<br><br />
[[File:IGKU0020.png|700px]]<br><br />
2. Confirm expression amount of GFP in E. coli that is activated other protein by IPTG and not activated E.coli as negative control and check whether expression amount of GFP depends on copy number<br><br />
[[File:IGKU0021.png|500px]]<br><br />
<br />
==Result==<br />
[[File:GuchaguchaFluorescenceProtein]]<br><br />
<br />
==Discussion==<br />
Thus, when you plate E. coli by usual method, the E. coli express GFP ununiformly. This is because you can not plate E. coli enough uniformly. As long as the expression of GFP is so ununiform, even if you set maximum area of cell-unit which is necessary to generate pattern on a plate, the gaps of average mass of GFP expression between cell-units are large enough. When you set an enough small area to generate pattern, you should plate so uniformly that you can consider the gap of mass of GFP expression between cell-units are small enough. So it is necessary to refine the plating method. For that, wet lab should plate many times, dry lab should analyze the results every time, reach the minimum area of cell-unit which we can consider the gap of average mass of GFP expression small enough, and provide the dates for we lab. And wet lab refine the method. Thus, if wet lab and dry lab understand enough and go some way along each other, you can construct more accurate and more reliable method.<br><br />
As we have seen, there are factor means E. coli density we must consider when we think intercellular system. On the other hand, when we think a system inside the cell, the factor E. coli density is unrelated and do not have to consider. Therefore, after this, we think system inside the cell.<br />
<br />
==Conclusion==<br />
As we showed the example ’Turing Pattern’, the results of wet lab and dry lab are often different because of their lack of understanding and appreciation each other. If both of them provide more information and closely discuss together, wet lab may be able to make an experimental system which imitates the system dry lab approximated to the real system. And wet lab provide quantified datas of a value which are necessary to formularize. If dry lab get these dates, they can create formulae which are well adapted to real system and run well simplified simulation. And if wet lab receive the anticipation date, they will be able to find more interesting results. When dry lab and wet lab join hands like this example ’Turing Pattern’, you can overthrow the future that some experiments should fail. Then, biology would evolve faster.<br />
<br />
==Reference==<br />
1:[http://www.sciencedirect.com/science/article/pii/S0092824005800084 A.M. Turing (1990) "The chemical basis of morphogenesis" Bulletin of Mathmatical Biology Vol. 52, No. 1/2, pp. 153-197]<br><br />
2:<html><a href="http://www.fbs.osaka-u.ac.jp/labs/skondo/paper/kondo%20IJDB%20review.pdf">S. Kondo et al(2009) "How animals get their skin patterns: fish pigment pattern as a live Turing wave" Int. J. Dev. Biol. 53: 851-856</a><br></html><br />
3:<html><a href="http://www.pnas.org/content/106/21/8429.short">Akiko Nakamasu et al(2009) "Interactions between zebrafish pigment cells responsible for the generation of Turing patterns" PNAS vol. 106 no. 21 8429–8434</a><br></html><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T01:02:02Z<p>Niina: /* Experiment */</p>
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=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
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<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
*Spinach-DT<br />
Experimental group<br><br />
*tetR aptamer-DT<br />
*antisense-spinach-DT<br />
*spinach-DT<br />
*and GFP generator<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab.<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
<br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T01:00:08Z<p>Niina: /* Experiment */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
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<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Attenuator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Activator===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br><br />
samples are following:<br><br />
Negative control<br><br />
Spinach-DT<br />
Experimental group<br><br />
tetR aptamer-DT<br />
antisense-spinach-DT<br />
spinach-DT<br />
and GFP generator<br><br />
We also check whether fusion RNA designed by us functions or not considering secondary structure<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab.<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[1][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[2][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[3][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[4][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
<br />
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:33:31Z<p>Niina: /* Result */</p>
<hr />
<div>{{Kyoto/header}}<br />
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<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
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<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Repressor===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab.<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:32:42Z<p>Niina: /* Future work */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Repressor===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br><br />
Then, after finishing construction of gene circuits that makes oscilation, we assay the oscilation circuit in wet lab.<br><br />
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:26:16Z<p>Niina: /* Future work */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
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<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
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<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Repressor===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br><br />
After that, we will substitute the values for oscilation model and try to solve simulate.<br><br />
And we will continue assaying of our parts<br><br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/ProjectTuringTeam:Kyoto/ProjectTuring2013-09-28T00:22:42Z<p>Niina: /* Introduction */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=Turing Model<br>-the problems between wet and dry-=<br />
==Introduction==<br />
On the Earth, there are various animals which have various patterns on their skin. The mechanism of this pattern formation has not been explained by any valid theories yet, although many hypothesis has been proposed. Among these hypothesis, there is a model pattern called Turing pattern proposed by A. Turing, a famous mathematician *1. S. Kondo *2 and some other researchers *3 suggests that some creatures’ pattern can be explained by Turing’s model. Here we will step by step explain how this Turing pattern is expressed by his model.<br><br />
[[File:IGKU0002.png|300px]]<br><br />
Let’s take a look on a simple hypothetical pattern formed by just two colors. Creatures’ epidermal pattern is expressed on the cells. Let’s assume that the pattern is formed by cells in different state α and β for example. A Cell in state α expresses color 1 and changes neighboring cells in state β into state α. Another Cell in state β expresses color 2 and changes neighboring cells in state α into state β, and remote cells in state β into state α. For convenience, hereafter we call the cell in state α {α}, and cell in the state β {β}.<br><br />
[[File:IGKU0003.png|200px]][[File:IGKU0004.png|400px]]<br><br />
Then, we will take a look on the system that two cells {&alpha;} and {&beta;} are existing uniformly and both of the cells are in the equilibrium state of interaction. Now suppose that the density of {&alpha;} and {&beta;} fluctuated in somewhere in the system. Assume that the density of cell {&beta;} increases like the cells in center of figure 1. At first, the center {&beta;} changes close {&alpha;} into {&beta;}. And next same {&beta;} changes remote {&beta;} into {&alpha;}. Then remote {&alpha;} changes close {&beta;} into {&alpha;}. The pattern is formed as this interaction continues.<br><br />
[[File:stripeform.gif]]<br><br />
Like this, a striped pattern is formed from close-and-remote interaction between two states of cell. Seeing this close-and-remote interaction separately, close interaction can be explained as positive feedback reaction in the aspects of polarization. Conversely, remote interaction can be explained as negative feedback reaction.<br><br />
[[File:IGKU0006.png|230px]][[File:IGKU0001.png|300px]]<br><br />
Diffusing substances such as proteins secreted from the cells determine the characters of the cells. It seems that the characters which changes close or remote cells (i.e. &alpha; and &beta;) are function of these diffusing substances. In other words, it can be said that {&alpha;} and {&beda;} secretes different diffusing substances, and the substances lead to close interaction (positive feedback) and remote interaction (negative feedback). Therefore, the pattern formation can be said to be formed by interaction between diffusible substances, as well as cell-cell interaction.<br><br />
[[File:IGKU0008.png|200px]]<br><br />
Then let’s consider about this interactions between diffusible substances in simplified model. Living organism’s body surface consists of cells shaped and sized ununiformly, therefore it is easier to understand if you assume that the body surface is a plane and consists of square cell-units sized uniformly. In this model, we can set diffusible substances which are secreted by {&alpha;} and {&beta;}, and they increase and decrease under the influence of interactions. And then, they are substances which has the same characteristic of cell {&alpha;} and {&beta;} (substances lead to close interaction (positive feedback) and remote interaction (negative feedback)), as a causative agent of the pattern formation on this model surfaces.<br><br />
[[File:IGKU0009.png|150px]][[File:IGKU0011.png|300px]]<br><br />
Then let’s have a look on the interaction between two diffusible substances; one lead to close interaction (positive feedback) and other lead to remote interaction (negative feedback). Hereafter we name this diffusible substances A and B. A has large diffusion velocity and represses B’s increase. B has the small diffusion velocity and promotes both A and B’s increase. If A and B has this characteristic, close interaction (positive feedback) and remote interaction (negative feedback) are formed. A and B forms legato density gradient due to this interaction. When each cell units have the character “Color the appropriate color answering to the denser one among the two diffusible substances inside the cell unit,” the substances’ density gradient can be imagined as pattern of cells.<br />
<br><br />
[[File:IGKU0010.png|500px]]<br><br />
Now let’s consider about how these two diffusible substances interacting each other in each cell unit. The amount of two diffusible substances in each cell unit changes only by diffusion and interaction. Then let’s focus on a certain cell unit (i) and consider about the concentration change. The substances amount change by diffusion is the difference between outflow and inflow. Change by the interaction is dependent on the amount of A and B at the certain moment. <br />
<br><br />
[[File:Hannou.png|400px]]<br><br />
[[File:Hannou2.png|300px]]<br />
<br><br />
Actually, this formula is the same as reaction-diffusion which is proposed by Turing for the purpose of explaining each factors of Turing pattern formation.<br />
It seems to be difficult to understand the content of these formulae. We’re going to explain the content.<br />
<br><br />
Ki, Ki’, and Ki’’ are the constant numbers which indicates how big the influence on interaction of each diffusible substances per unit quantity is. In other words, these terms returns the amount of A and B at a moment depending on the interaction from the amount of A and B at anterior moment. DiA and DiB are the constant numbers peculiar to each diffusible substances which indicates the tendency of diffusion of A and B here. In other words, the terms DiA and DiB is the superficial inflow-outflow budget depending on diffusion of A and B. This is the contents which is described by the equitation.<br />
<br><br />
[[File:IGKU0012.png|300px]][[File:IGKU0013.png|200px]]<br><br />
[[File:IGKU0014.png|300px]][[File:IGKU0015.png|300px]]<br><br />
<br><br />
<br />
==Experiments==<br />
We focused on the constants "Ki, Ki’, Ki’’" in these formulas. These are took as a given as "always fixed in any point" to Turing pattern. However, in fact, is it true that Ki is always fixed in any point with Turing pattern formed by E. coli? We thought it is not always true in wet work because E. coli makes A and B. In other words, increase or decrease speed of amount of A and B in a certain point depends on E. coli dencity in the point.<br><br />
As long as E. coli is growing not uniformly until a steady state, it must be generated E. coli density difference between each point. This E.coli density difference makes "Ki, Ki’, Ki’’" change between each point.<br><br />
Can we ignored "Ki, Ki’, Ki’’" difference? To confirm this, we established these assay.<br><br><br />
1. Confirm expression amount of GFP in both a steady state and a non-steady state with plated E. coli by common method.<br><br />
[[File:IGKU0020.png|700px]]<br><br />
2. Confirm expression amount of GFP in E. coli that is activated other protein by IPTG and not activated E.coli as negative control and check whether expression amount of GFP depends on copy number<br><br />
[[File:IGKU0021.png|500px]]<br><br />
<br />
==Result==<br />
<br />
<br />
==Discussion==<br />
Thus, when you plate E. coli by usual method, the E. coli express GFP ununiformly. This is because you can not plate E. coli enough uniformly. As long as the expression of GFP is so ununiform, even if you set maximum area of cell-unit which is necessary to generate pattern on a plate, the gaps of average mass of GFP expression between cell-units are large enough. When you set an enough small area to generate pattern, you should plate so uniformly that you can consider the gap of mass of GFP expression between cell-units are small enough. So it is necessary to refine the plating method. For that, wet lab should plate many times, dry lab should analyze the results every time, reach the minimum area of cell-unit which we can consider the gap of average mass of GFP expression small enough, and provide the dates for we lab. And wet lab refine the method. Thus, if wet lab and dry lab understand enough and go some way along each other, you can construct more accurate and more reliable method.<br><br />
As we have seen, there are factor means E. coli density we must consider when we think intercellular system. On the other hand, when we think a system inside the cell, the factor E. coli density is unrelated and do not have to consider. Therefore, after this, we think system inside the cell.<br />
<br />
==Conclusion==<br />
As we showed the example ’Turing Pattern’, the results of wet lab and dry lab are often different because of their lack of understanding and appreciation each other. If both of them provide more information and closely discuss together, wet lab may be able to make an experimental system which imitates the system dry lab approximated to the real system. And wet lab provide quantified datas of a value which are necessary to formularize. If dry lab get these dates, they can create formulae which are well adapted to real system and run well simplified simulation. And if wet lab receive the anticipation date, they will be able to find more interesting results. When dry lab and wet lab join hands like this example ’Turing Pattern’, you can overthrow the future that some experiments should fail. Then, biology would evolve faster.<br />
<br />
==Reference==<br />
1:[http://www.sciencedirect.com/science/article/pii/S0092824005800084 A.M. Turing (1990) "The chemical basis of morphogenesis" Bulletin of Mathmatical Biology Vol. 52, No. 1/2, pp. 153-197]<br><br />
2:<html><a href="http://www.fbs.osaka-u.ac.jp/labs/skondo/paper/kondo%20IJDB%20review.pdf">S. Kondo et al(2009) "How animals get their skin patterns: fish pigment pattern as a live Turing wave" Int. J. Dev. Biol. 53: 851-856</a><br></html><br />
3:<html><a href="http://www.pnas.org/content/106/21/8429.short">Akiko Nakamasu et al(2009) "Interactions between zebrafish pigment cells responsible for the generation of Turing patterns" PNAS vol. 106 no. 21 8429–8434</a><br></html><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:16:14Z<p>Niina: /* Future Work */</p>
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
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<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
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<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Repressor===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:15:39Z<p>Niina: /* 文章のたまり場 */</p>
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<html><br />
<center><br />
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</html><br />
<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Repressor===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:15:16Z<p>Niina: /* future work */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
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<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Repressor===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==Future work==<br />
To solve simultaneous differential equations meaning oscilation model numerically, we will try to found exact values of some constants. For example, to determine binding constant between tetR and tetR aptamer, we will try to build up assay method and to get quantitative data.<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:06:46Z<p>Niina: /* Achievement */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br />
<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Repressor===<br />
We took up TetR aptamer as an example of functional RNA which activates a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:05:58Z<p>Niina: /* Oscillation */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
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<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
【メモ:Assay、Result、Discussion】<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
===Achievement===<br />
我々は、このプロジェクトで以下のことを達成した。<br><br />
①<br><br />
②<br><br />
③<br><br />
④<br><br />
⑤<br><br />
⑥<br><br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-28T00:05:43Z<p>Niina: /* Future Work */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
細胞間の相互作用のシミュレーションは、細胞内の条件だけでなく位置関係などさらに複雑な条件を考慮しなければならないため、かなり複雑で手におえない<br />
そこで1細胞内でのシミュレーションにおいて、dryとwetの解離の対策を考えてみた。合成生物学のある研究(a fast robustなんとかかんとか)[citation*]では、オシレーションの形成をdryでもwetでも確認している。この実験系では、細胞分裂という、オシレーションの形成に強く影響を与えるであろう要素をゼロに近似できるようなロバストな回路がコンストラクトされている。このことから、dryでwetの系を考慮しきるのが難しいことの1つの解決法として、適切な近似計算によって複雑性を無視できるロバストネスを持った回路を構築することが考えられる。しかしそのような構成要素はそう多くなく、適応する系は限られている。そこで私たちは押しレーションの構築を他のアプローチによって実現し、dryに歩み寄るwetの系を考えてみた。私たちはrnaを構成要素とする押しレーションの構築をゴールとした。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Oscillation===<br />
その回路として、ncRNA-mRNAの相互作用を抑制機構に持ち、RNA アプタマーとtetR Proteinを促進機構に用いた以下の様な回路を提案する。アウトプット機構としては、Spinachを想定する。<br />
<br />
<prezi><br />
<br />
This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator.<br />
<br />
促進、抑制の各機構と、この回路のアウトプットとしてのSpinachの機能は以下に述べる。<br />
<br />
<br />
</div><br />
<div id="reportertab"><br />
<br />
===Repressor===<br />
We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. ncRNA in pT181 plasmid (pT181 attenuator) controls the fate of transcriptional elongation in response to an input by complementary antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structure. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, attenuator region RNA folds into an alternative structure which allow transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA without other small molecules. Synthetic biologists variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). In this paper, many variants ofpT181 attenuator/antisense is constructed and the attenuation rate of each variants is different. We chose this mechanism in gene repression. <br />
</div><br />
<div id="repressiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
【メモ:Assay、Result、Discussion】<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
===Achievement===<br />
我々は、このプロジェクトで以下のことを達成した。<br><br />
①<br><br />
②<br><br />
③<br><br />
④<br><br />
⑤<br><br />
⑥<br><br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[x][http://www.nature.com/nature/journal/v456/n7221/abs/nature07389.html Jesse Stricker et al.(2008)"A fast, robust and tunable synthetic gene oscillator" Nature 456, 516-519]<br><br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-27T23:57:23Z<p>Niina: /* Conclusion */</p>
<hr />
<div>{{Kyoto/header}}<br />
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<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
<p>先のチューリングの例でみたとおり、場のスケールを多數の大腸菌で考えると菌体密度のようなfactorが影響してきてしまう。そのため、反応が複雑になってしまい、wetとdryの乖離を進める一因となっている。そこで、主要なファクターを考えやすくするために、場のスケールを一細胞で考えようと試みた。ここで扱うモデルは、よく研究されているオシレーションである。</p><br />
<p> As we state in Turing Model Project, if we try to make turing pattern with many cells of E.coli, we have to consider irregular factors such as density of cells. So, we have to simulate too complex circle to be practical. This complexity makes wet and dry unmatchable. So, then, we tried to compose turing pattern model on the inside of each E.coli’s cell. We adopted well-researched oscillation model. </p><br />
<br />
細胞内のオシレーションとしては、例えばシアノバクテリアのkai protein familyなどがある<reference>。Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。しかし、kaiのオシレーションを大腸菌で実現しようとすると。周期の時間がスケールが大腸菌の分裂速度よりも大幅に違う。よって、正確にモデリングすることが困難だろう。だから、短時間のモデルが考えやすいだろう。<br><br />
<p> In the natural world, for example, kai protein family oscillates in the cell of cyanobacteria. <reference> Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。However, because the cycle of kai protein family’s oscillation is much longer than E.coli’s cell cycle, it is difficult to create precise model of kai protein in E.coli. Hence, quicker oscillation is easy to simulate.</p><br />
<br />
さて、チューリングのモデルを大腸菌内で、かつ短い時間スケールで実現する際のfactorとして、私たちはRNAを提唱する。実際、転写調節因子としてのRNAの研究は始まっている<reference>。RNAを使う利点としては、次の2点が挙げられる。<br><br />
<p> Then, as quickly-oscillating factor in E.coli, we advocate functional RNA. In fact, research of RNA to regulate transcription have been undertaken. <reference> The merits of adopting functional RNA is following:</p><br />
<br />
<引用>4RNAは二次構造の予測や、RNA同士やDNAに対する特異的な結合を可能にするような設計を行うこともタンパク質に比較すると容易である。よって、遺伝子回路を製作するにあたって、回路を構成するRNA同士が塩基配列特異的な相互作用をするように設計すれば、数に限りがある既存のアクチベーターやリプレッサータンパク質を用いては不可能だったような、一細胞内で複数の独立した回路を共存させるということが可能になる。加えて、回路に直接関係しない任意の遺伝子の発現量をそれ同調させることも可能となる。<br />
<br />
Firstly, compared to protein, it is easier to predict the secondary structure of RNA, and to design the structure in order to bind to a specific RNA or DNA. Therefore, if we design the RNA which constructs the circuit to interact specifically to the base sequence, we can make some different circuits co-exist inside one cell. Moreover, since we can predict the structure we can link a post-transcriptional RNA reporter to the functional RNA to stop the conformational alternation, and realize an imaging of the RNA.<br />
<br />
5さらにRNAは転写後、機能するまでに翻訳の時間を要しないため、応答までの時間が短縮される。また、生体内での分解もタンパク質と比較して早いので、転写調節から応答までの時間を比較的短くすることも可能になると考えられる。そのため、遺伝子回路を構成する分子を決定するとき、 タンパク質とRNAを適宜織り交ぜることで、転写調節から目的分子の細胞内の量を調節する時間をより広い幅でcontrolできるようになるかもしれない。</引用><br />
<br />
今回、私たちの用いるオシレーションのモデルは、<チューリングのアレ>です。このモデルにはactivatorとrepressorが必要です。それぞれの要素については以下に述べる。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Repressor===<br />
<p>We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. Some kinds of ncRNA work as a transcriptional repressor in vivo, for example, Gram-negative bacteria <i>Staphylococcus aureus</i> regulates a copy number of plasmid called pT181 in this mechanism.<sup>5</sup>The ncRNA in pT181 plasmid controls the fate of transcriptional elongation in response to an input by antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structures. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, RNA in attenuator region folds into an alternative structure which allows transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA and without other small molecules, many synthetic biologist constructed a variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). We chose this mechanism in gene repression. </p><br />
[[Image: 2013IGKUprojectRNArepressionMECHANISM.png]][[Image:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
<p>To ensure the function of antisense RNA and attenuator region, we will compare the amount of mRNA of GFP located in the downstream of an attenuator region in the presence and absence of antisense RNA.</p><br />
<br />
<p>転写の抑制を行うようなRNAの例として、我々は、伝令RNAに相補的に結合するncRNAによる転写制御を挙げる。これは、生体内でのRNAによるゲノム転写機構のひとつ、Gram-negative bacteria Staphylococcus aureusのpT181と呼ばれるplasmidなどのコピー数のregulationの機構である。RepressorとなるRNA (Antisense RNA)がある状態では、プロモーター下流のAttenuator locusがRho-independent terminator を形成することによりgenome coding部位の転写が抑制されるが、if the antisense RNA fails to bind, nascent RNA refolds into an alternative structure which prevents termination and promotes read-through (Novick, 1989) という仕組みを用いている。この機構は、他のリボスイッチと違いRNAのみで他の低分子化合物を用いていないため、合成生物学の新たな手法として、塩基置換などにより様々なタイプのものが作られている (Takahashi et al, 2013)。<br />
われわれはこれをRepressionの回路とした。AttenuatorとAntisenseの、Attenuator Regionより下流の遺伝子の転写を阻害する機能を確認するため、Attenuator antisense RNAの存在下と非存在下で、Attenuator Region下流のGFP遺伝子の発現量を比較した。 </p><br />
<p> In order to check the function of Attenuator and Antisense, we introduced Attenuator and Antisense into E.coli as experimental groups. We compared this E.coli with several controlled group in expression of GFP.</p><br />
<p>------const------</p><br />
Positive Control<br><br />
A-B: Pcon-RNAs(tetRaptamer, attenuator), Pcon-atte-GFP<br><br />
-antisenseを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、antisenseのもつ相補的配列が問題であることを確かめる。<br><br />
C: Pcon-atte-GFP<br><br />
Antisense非存在下においてはGFPの発現は抑制されないことを確認する。<br><br />
Experimental Group<br><br />
D: Pcon-antisense Pcon-atte-GFP<br><br />
Positive Control<br><br />
<br><br />
A-B. Pcon-RNAs(tetRaptamer, attenuator), Pcon-attenuator-GFP<br><br />
<p>In order to check the uniqueness of Antisense in repression, we introduced other RNAs into E.coli.</p><br />
C. Pcon-attenuator-GFP<br><br />
<p>This E.coli shows that if there is no Antisense, the expression of GFP is not repressed.</p><br />
Experimental Group<br><br />
D. Pcon-antisense Pcon-attenuator-GFP<br><br />
[[Image: IGKUprojectRNArepressionCONST2.png]]<br />
<p>------const------</p><br />
<p>---figcaption----</p><br />
<p> Antisenseが常時発現している大腸菌(figE)においてはAttenuator Regionの下流にあるGFPの転写が抑制され、Antisenseが存在しない大腸菌(figD)では抑制されていないことから、figEの大腸菌における転写抑制はAntisenseに起因することがわかる。figEの大腸菌でAntisenseをコードしていた部分を他の配列に置き換えた大腸菌(figA-C)におけるGFPの転写量はAntisenseを転写しない大腸菌(figD)に比べて遜色ないことから、figEの大腸菌での転写抑制はAtternatorに特異的なものであったことが導かれる。 </p><br />
<p> Compared with E.coli which didn’t express Antisense(figC), E.coli which always expresses Antisense(figD) was repressed in expression of GFP. This demonstrates that this repression was caused by expression of Antisense. Furthermore, because E.coli in which other structure of RNA was introduced(figA-B) expresses as much GFP as E.coli which didn’t expresses Antisense, we can say that this repression was peculiar to Antisense. </p><br />
<p>---figcaption----</p><br />
</div><br />
<div id="reportertab"><br />
<br />
===Activator===<br />
<p>We pick up tetR aptamer as an example of functional RNA which activates transcription. TetR aptamer specifically binds tet represser (tetR), which binds DNA specific site, repress transcription of downstream gene, and induce tetR conformational change and tetR reorientation.<sup>7</sup> That is, if in one cell, tetR is constantly expressed, gene located in the downstream of tet promoter is usually repressed and only when tetR aptamer is being expressed, it derepressed and transcribed. </p><br />
[[Image: 2013IGKUprojectRNAactivationMECHANISM.png]]<br />
<p>In our experiment, we check and measure tetR aptamer’s function in E.coli by comparing GFP fluorescence regulated by tetR promoter and GFP expression level by qRT-PCR in the following 4 cellular cases:1 TetR and tetR aptamer is constantly expressed. 2 Only tetR is constantly expressed and tetR aptamer is not induced.3 TetR and other functional RNA is constantly expressed. 4 TetR is not induced.</p><br />
<br />
<p>転写のアクチベーションを行うような機能性RNAの例として、我々はtetR aptamerを挙げる。これはtet repressorに特異的に結合するアプタマーであるが、DNAの特定領域に結合して転写を抑制しているtet repressorに結合してDNAから解離させる作用も持つ。つまり、常に一定量のtet repressorが発現し、存在しているような細胞内では、tetR aptamerが発現している間のみtet promotor以下の転写の抑制が解除、つまり活性化され、tetR aptamerが発現していず存在していない場合は、tetRの機能によって転写が抑制されるようになる。 tetR aptamerの働きを確認するため、tetRタンパク質とtetR aptamerを常時発現させた場合と、tetRタンパク質のみを常時発現させた場合、tetRとtetR アプタマー以外の構造をもつRNAを発現させた場合、tetRを発現させなかった場合とで、tetプロモーター下流に配置したGFP遺伝子を発現させその蛍光を見、qRT-PCRで発現量を比較しtetR aptamerの働きを確認した。(顕微鏡で蛍光度の差が確認できたときはqRT-PCRは補強扱いとし、確認できなかった場合はqRT-PCRのみを蛍光度の比較の尺度とする。) </p><br />
<p>In order to check the function of tetRaptamer, we introduced tetR and tetRaptamer as experimental group. We compared this E.coli with several controlled groups in expression of GFP.</p><br />
<p>-----コンストラクション------</p><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
-tetRを導入せず、Ptet-GFP単体のもの。tetRが存在しない場合にPtetがonになるということの確認。<br><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-TetR<br><br />
-tetRaptamerが存在しない場合。tetRがそのままで転写抑制をすることの確認。<br><br />
C-D. Ptet-GFP, Pcon-TetR, Pcon-RNAs(anti_attenuator, attenuator)<br><br />
-tetR aptamerを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、tetR aptamerのみが持つ構造と機能が問題であることを確かめる。<br><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-TetR, Pcon-tetRaptamer<br><br />
<br><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
<p>In order to check whether the GFP gene is expressed when there is no tetR protein, we introduced only Ptet-GFP.</p><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-tetR<br><br />
<p>Through this E.coli, it is confirmed that when tetR is expressed, tetR surpresses the expression of GFP.</p><br />
C-D. Ptet-GFP, Pcon-tetR, Pcon-RNAs(antisense, attenuator)<br><br />
<p>These kinds of E.coli shows that the surpression of the function of tetR is caused only by tetRaptamer.</p><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-tetR, Pcon-tetRaptamer<br><br />
[[Image: 2013IGKUprojectRNAactivatorCONST.png]]<br />
<p>-----コンストラクション------</p><br />
<p>-----fig Caption------</p><br />
<p> tetRを発現しない大腸菌(figA)の蛍光はtetRを発現する大腸菌(figB)のそれよりも強いことから、tetRはtetリプレッサーに結合して下流の転写を妨げることがわかる。tetRを発現し、tetRaptamerを転写しない大腸菌(figB)のGFP転写量に比べてtetRとtetRaptamerの両方を発現する大腸菌(figF)のGFP転写量が有意に大きいことから、tetRaptamerはtetRによる転写の抑制を解除する働きがあることが示唆される。tetRaptamerをコードしていた部分を他の配列に置き換えた大腸菌(figC-E)の蛍光はtetRとtetRaptamerの両方を発現する大腸菌(figF)よりも弱く、tetRのみを発現する大腸菌(figB)と同程度であることから、figFの大腸菌におけるtetRの機能の抑制はtetRaptamerに特有のものであることがわかる。</p><br />
<p> The fact that the fluorescence of E.coli which expressed tetR(figB) was weaker than E.coli which didn’t express tetR(figA) shows that tetR represses the expression of genes at the downstream of tet promotor. Since E.coli introduced tetR and tetRaptamer (figF) expressed more GFP than E.coli introduced only tetR(figB), we were confirmed that tetRaptamer cancels the effect of tetR {in some degree / almost completely}. Moreover, E.coli which was introduced other structure of RNA(figC-E) could express as much GFP as E.coli introduced tetR only(figB), which demonstrates that the surpression of the function of tetR protein is unique to tetRaptamer. </p><br />
<p>-----fig Caption------</p><br />
</div><br />
<div id="repressiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
【メモ:Assay、Result、Discussion】<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
We got ready for construction oscilator circuit in wet lab.<br><br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<html><br />
<center><br />
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<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
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<div id="achievetab"><br />
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===Achievement===<br />
我々は、このプロジェクトで以下のことを達成した。<br><br />
①<br><br />
②<br><br />
③<br><br />
④<br><br />
⑤<br><br />
⑥<br><br />
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<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
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<div id="referencetab"><br />
<br />
== Reference ==<br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
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{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-27T23:52:49Z<p>Niina: /* Conclusion */</p>
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<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
<p>先のチューリングの例でみたとおり、場のスケールを多數の大腸菌で考えると菌体密度のようなfactorが影響してきてしまう。そのため、反応が複雑になってしまい、wetとdryの乖離を進める一因となっている。そこで、主要なファクターを考えやすくするために、場のスケールを一細胞で考えようと試みた。ここで扱うモデルは、よく研究されているオシレーションである。</p><br />
<p> As we state in Turing Model Project, if we try to make turing pattern with many cells of E.coli, we have to consider irregular factors such as density of cells. So, we have to simulate too complex circle to be practical. This complexity makes wet and dry unmatchable. So, then, we tried to compose turing pattern model on the inside of each E.coli’s cell. We adopted well-researched oscillation model. </p><br />
<br />
細胞内のオシレーションとしては、例えばシアノバクテリアのkai protein familyなどがある<reference>。Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。しかし、kaiのオシレーションを大腸菌で実現しようとすると。周期の時間がスケールが大腸菌の分裂速度よりも大幅に違う。よって、正確にモデリングすることが困難だろう。だから、短時間のモデルが考えやすいだろう。<br><br />
<p> In the natural world, for example, kai protein family oscillates in the cell of cyanobacteria. <reference> Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。However, because the cycle of kai protein family’s oscillation is much longer than E.coli’s cell cycle, it is difficult to create precise model of kai protein in E.coli. Hence, quicker oscillation is easy to simulate.</p><br />
<br />
さて、チューリングのモデルを大腸菌内で、かつ短い時間スケールで実現する際のfactorとして、私たちはRNAを提唱する。実際、転写調節因子としてのRNAの研究は始まっている<reference>。RNAを使う利点としては、次の2点が挙げられる。<br><br />
<p> Then, as quickly-oscillating factor in E.coli, we advocate functional RNA. In fact, research of RNA to regulate transcription have been undertaken. <reference> The merits of adopting functional RNA is following:</p><br />
<br />
<引用>4RNAは二次構造の予測や、RNA同士やDNAに対する特異的な結合を可能にするような設計を行うこともタンパク質に比較すると容易である。よって、遺伝子回路を製作するにあたって、回路を構成するRNA同士が塩基配列特異的な相互作用をするように設計すれば、数に限りがある既存のアクチベーターやリプレッサータンパク質を用いては不可能だったような、一細胞内で複数の独立した回路を共存させるということが可能になる。加えて、回路に直接関係しない任意の遺伝子の発現量をそれ同調させることも可能となる。<br />
<br />
Firstly, compared to protein, it is easier to predict the secondary structure of RNA, and to design the structure in order to bind to a specific RNA or DNA. Therefore, if we design the RNA which constructs the circuit to interact specifically to the base sequence, we can make some different circuits co-exist inside one cell. Moreover, since we can predict the structure we can link a post-transcriptional RNA reporter to the functional RNA to stop the conformational alternation, and realize an imaging of the RNA.<br />
<br />
5さらにRNAは転写後、機能するまでに翻訳の時間を要しないため、応答までの時間が短縮される。また、生体内での分解もタンパク質と比較して早いので、転写調節から応答までの時間を比較的短くすることも可能になると考えられる。そのため、遺伝子回路を構成する分子を決定するとき、 タンパク質とRNAを適宜織り交ぜることで、転写調節から目的分子の細胞内の量を調節する時間をより広い幅でcontrolできるようになるかもしれない。</引用><br />
<br />
今回、私たちの用いるオシレーションのモデルは、<チューリングのアレ>です。このモデルにはactivatorとrepressorが必要です。それぞれの要素については以下に述べる。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Repressor===<br />
<p>We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. Some kinds of ncRNA work as a transcriptional repressor in vivo, for example, Gram-negative bacteria <i>Staphylococcus aureus</i> regulates a copy number of plasmid called pT181 in this mechanism.<sup>5</sup>The ncRNA in pT181 plasmid controls the fate of transcriptional elongation in response to an input by antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structures. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, RNA in attenuator region folds into an alternative structure which allows transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA and without other small molecules, many synthetic biologist constructed a variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). We chose this mechanism in gene repression. </p><br />
[[Image: 2013IGKUprojectRNArepressionMECHANISM.png]][[Image:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
<p>To ensure the function of antisense RNA and attenuator region, we will compare the amount of mRNA of GFP located in the downstream of an attenuator region in the presence and absence of antisense RNA.</p><br />
<br />
<p>転写の抑制を行うようなRNAの例として、我々は、伝令RNAに相補的に結合するncRNAによる転写制御を挙げる。これは、生体内でのRNAによるゲノム転写機構のひとつ、Gram-negative bacteria Staphylococcus aureusのpT181と呼ばれるplasmidなどのコピー数のregulationの機構である。RepressorとなるRNA (Antisense RNA)がある状態では、プロモーター下流のAttenuator locusがRho-independent terminator を形成することによりgenome coding部位の転写が抑制されるが、if the antisense RNA fails to bind, nascent RNA refolds into an alternative structure which prevents termination and promotes read-through (Novick, 1989) という仕組みを用いている。この機構は、他のリボスイッチと違いRNAのみで他の低分子化合物を用いていないため、合成生物学の新たな手法として、塩基置換などにより様々なタイプのものが作られている (Takahashi et al, 2013)。<br />
われわれはこれをRepressionの回路とした。AttenuatorとAntisenseの、Attenuator Regionより下流の遺伝子の転写を阻害する機能を確認するため、Attenuator antisense RNAの存在下と非存在下で、Attenuator Region下流のGFP遺伝子の発現量を比較した。 </p><br />
<p> In order to check the function of Attenuator and Antisense, we introduced Attenuator and Antisense into E.coli as experimental groups. We compared this E.coli with several controlled group in expression of GFP.</p><br />
<p>------const------</p><br />
Positive Control<br><br />
A-B: Pcon-RNAs(tetRaptamer, attenuator), Pcon-atte-GFP<br><br />
-antisenseを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、antisenseのもつ相補的配列が問題であることを確かめる。<br><br />
C: Pcon-atte-GFP<br><br />
Antisense非存在下においてはGFPの発現は抑制されないことを確認する。<br><br />
Experimental Group<br><br />
D: Pcon-antisense Pcon-atte-GFP<br><br />
Positive Control<br><br />
<br><br />
A-B. Pcon-RNAs(tetRaptamer, attenuator), Pcon-attenuator-GFP<br><br />
<p>In order to check the uniqueness of Antisense in repression, we introduced other RNAs into E.coli.</p><br />
C. Pcon-attenuator-GFP<br><br />
<p>This E.coli shows that if there is no Antisense, the expression of GFP is not repressed.</p><br />
Experimental Group<br><br />
D. Pcon-antisense Pcon-attenuator-GFP<br><br />
[[Image: IGKUprojectRNArepressionCONST2.png]]<br />
<p>------const------</p><br />
<p>---figcaption----</p><br />
<p> Antisenseが常時発現している大腸菌(figE)においてはAttenuator Regionの下流にあるGFPの転写が抑制され、Antisenseが存在しない大腸菌(figD)では抑制されていないことから、figEの大腸菌における転写抑制はAntisenseに起因することがわかる。figEの大腸菌でAntisenseをコードしていた部分を他の配列に置き換えた大腸菌(figA-C)におけるGFPの転写量はAntisenseを転写しない大腸菌(figD)に比べて遜色ないことから、figEの大腸菌での転写抑制はAtternatorに特異的なものであったことが導かれる。 </p><br />
<p> Compared with E.coli which didn’t express Antisense(figC), E.coli which always expresses Antisense(figD) was repressed in expression of GFP. This demonstrates that this repression was caused by expression of Antisense. Furthermore, because E.coli in which other structure of RNA was introduced(figA-B) expresses as much GFP as E.coli which didn’t expresses Antisense, we can say that this repression was peculiar to Antisense. </p><br />
<p>---figcaption----</p><br />
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<div id="reportertab"><br />
<br />
===Activator===<br />
<p>We pick up tetR aptamer as an example of functional RNA which activates transcription. TetR aptamer specifically binds tet represser (tetR), which binds DNA specific site, repress transcription of downstream gene, and induce tetR conformational change and tetR reorientation.<sup>7</sup> That is, if in one cell, tetR is constantly expressed, gene located in the downstream of tet promoter is usually repressed and only when tetR aptamer is being expressed, it derepressed and transcribed. </p><br />
[[Image: 2013IGKUprojectRNAactivationMECHANISM.png]]<br />
<p>In our experiment, we check and measure tetR aptamer’s function in E.coli by comparing GFP fluorescence regulated by tetR promoter and GFP expression level by qRT-PCR in the following 4 cellular cases:1 TetR and tetR aptamer is constantly expressed. 2 Only tetR is constantly expressed and tetR aptamer is not induced.3 TetR and other functional RNA is constantly expressed. 4 TetR is not induced.</p><br />
<br />
<p>転写のアクチベーションを行うような機能性RNAの例として、我々はtetR aptamerを挙げる。これはtet repressorに特異的に結合するアプタマーであるが、DNAの特定領域に結合して転写を抑制しているtet repressorに結合してDNAから解離させる作用も持つ。つまり、常に一定量のtet repressorが発現し、存在しているような細胞内では、tetR aptamerが発現している間のみtet promotor以下の転写の抑制が解除、つまり活性化され、tetR aptamerが発現していず存在していない場合は、tetRの機能によって転写が抑制されるようになる。 tetR aptamerの働きを確認するため、tetRタンパク質とtetR aptamerを常時発現させた場合と、tetRタンパク質のみを常時発現させた場合、tetRとtetR アプタマー以外の構造をもつRNAを発現させた場合、tetRを発現させなかった場合とで、tetプロモーター下流に配置したGFP遺伝子を発現させその蛍光を見、qRT-PCRで発現量を比較しtetR aptamerの働きを確認した。(顕微鏡で蛍光度の差が確認できたときはqRT-PCRは補強扱いとし、確認できなかった場合はqRT-PCRのみを蛍光度の比較の尺度とする。) </p><br />
<p>In order to check the function of tetRaptamer, we introduced tetR and tetRaptamer as experimental group. We compared this E.coli with several controlled groups in expression of GFP.</p><br />
<p>-----コンストラクション------</p><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
-tetRを導入せず、Ptet-GFP単体のもの。tetRが存在しない場合にPtetがonになるということの確認。<br><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-TetR<br><br />
-tetRaptamerが存在しない場合。tetRがそのままで転写抑制をすることの確認。<br><br />
C-D. Ptet-GFP, Pcon-TetR, Pcon-RNAs(anti_attenuator, attenuator)<br><br />
-tetR aptamerを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、tetR aptamerのみが持つ構造と機能が問題であることを確かめる。<br><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-TetR, Pcon-tetRaptamer<br><br />
<br><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
<p>In order to check whether the GFP gene is expressed when there is no tetR protein, we introduced only Ptet-GFP.</p><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-tetR<br><br />
<p>Through this E.coli, it is confirmed that when tetR is expressed, tetR surpresses the expression of GFP.</p><br />
C-D. Ptet-GFP, Pcon-tetR, Pcon-RNAs(antisense, attenuator)<br><br />
<p>These kinds of E.coli shows that the surpression of the function of tetR is caused only by tetRaptamer.</p><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-tetR, Pcon-tetRaptamer<br><br />
[[Image: 2013IGKUprojectRNAactivatorCONST.png]]<br />
<p>-----コンストラクション------</p><br />
<p>-----fig Caption------</p><br />
<p> tetRを発現しない大腸菌(figA)の蛍光はtetRを発現する大腸菌(figB)のそれよりも強いことから、tetRはtetリプレッサーに結合して下流の転写を妨げることがわかる。tetRを発現し、tetRaptamerを転写しない大腸菌(figB)のGFP転写量に比べてtetRとtetRaptamerの両方を発現する大腸菌(figF)のGFP転写量が有意に大きいことから、tetRaptamerはtetRによる転写の抑制を解除する働きがあることが示唆される。tetRaptamerをコードしていた部分を他の配列に置き換えた大腸菌(figC-E)の蛍光はtetRとtetRaptamerの両方を発現する大腸菌(figF)よりも弱く、tetRのみを発現する大腸菌(figB)と同程度であることから、figFの大腸菌におけるtetRの機能の抑制はtetRaptamerに特有のものであることがわかる。</p><br />
<p> The fact that the fluorescence of E.coli which expressed tetR(figB) was weaker than E.coli which didn’t express tetR(figA) shows that tetR represses the expression of genes at the downstream of tet promotor. Since E.coli introduced tetR and tetRaptamer (figF) expressed more GFP than E.coli introduced only tetR(figB), we were confirmed that tetRaptamer cancels the effect of tetR {in some degree / almost completely}. Moreover, E.coli which was introduced other structure of RNA(figC-E) could express as much GFP as E.coli introduced tetR only(figB), which demonstrates that the surpression of the function of tetR protein is unique to tetRaptamer. </p><br />
<p>-----fig Caption------</p><br />
</div><br />
<div id="repressiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
【メモ:Assay、Result、Discussion】<br />
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<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br><br />
We predicted second structure of fusion RNA: atenuator-tetRaptamer and antisence-spinach with centroid fold. It seems to be expected structure and to function as expected.<br><br />
wetで事実作れる準備ができた<br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
===Achievement===<br />
我々は、このプロジェクトで以下のことを達成した。<br><br />
①<br><br />
②<br><br />
③<br><br />
④<br><br />
⑤<br><br />
⑥<br><br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-27T23:44:13Z<p>Niina: /* RT-PCR */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
<p>先のチューリングの例でみたとおり、場のスケールを多數の大腸菌で考えると菌体密度のようなfactorが影響してきてしまう。そのため、反応が複雑になってしまい、wetとdryの乖離を進める一因となっている。そこで、主要なファクターを考えやすくするために、場のスケールを一細胞で考えようと試みた。ここで扱うモデルは、よく研究されているオシレーションである。</p><br />
<p> As we state in Turing Model Project, if we try to make turing pattern with many cells of E.coli, we have to consider irregular factors such as density of cells. So, we have to simulate too complex circle to be practical. This complexity makes wet and dry unmatchable. So, then, we tried to compose turing pattern model on the inside of each E.coli’s cell. We adopted well-researched oscillation model. </p><br />
<br />
細胞内のオシレーションとしては、例えばシアノバクテリアのkai protein familyなどがある<reference>。Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。しかし、kaiのオシレーションを大腸菌で実現しようとすると。周期の時間がスケールが大腸菌の分裂速度よりも大幅に違う。よって、正確にモデリングすることが困難だろう。だから、短時間のモデルが考えやすいだろう。<br><br />
<p> In the natural world, for example, kai protein family oscillates in the cell of cyanobacteria. <reference> Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。However, because the cycle of kai protein family’s oscillation is much longer than E.coli’s cell cycle, it is difficult to create precise model of kai protein in E.coli. Hence, quicker oscillation is easy to simulate.</p><br />
<br />
さて、チューリングのモデルを大腸菌内で、かつ短い時間スケールで実現する際のfactorとして、私たちはRNAを提唱する。実際、転写調節因子としてのRNAの研究は始まっている<reference>。RNAを使う利点としては、次の2点が挙げられる。<br><br />
<p> Then, as quickly-oscillating factor in E.coli, we advocate functional RNA. In fact, research of RNA to regulate transcription have been undertaken. <reference> The merits of adopting functional RNA is following:</p><br />
<br />
<引用>4RNAは二次構造の予測や、RNA同士やDNAに対する特異的な結合を可能にするような設計を行うこともタンパク質に比較すると容易である。よって、遺伝子回路を製作するにあたって、回路を構成するRNA同士が塩基配列特異的な相互作用をするように設計すれば、数に限りがある既存のアクチベーターやリプレッサータンパク質を用いては不可能だったような、一細胞内で複数の独立した回路を共存させるということが可能になる。加えて、回路に直接関係しない任意の遺伝子の発現量をそれ同調させることも可能となる。<br />
<br />
Firstly, compared to protein, it is easier to predict the secondary structure of RNA, and to design the structure in order to bind to a specific RNA or DNA. Therefore, if we design the RNA which constructs the circuit to interact specifically to the base sequence, we can make some different circuits co-exist inside one cell. Moreover, since we can predict the structure we can link a post-transcriptional RNA reporter to the functional RNA to stop the conformational alternation, and realize an imaging of the RNA.<br />
<br />
5さらにRNAは転写後、機能するまでに翻訳の時間を要しないため、応答までの時間が短縮される。また、生体内での分解もタンパク質と比較して早いので、転写調節から応答までの時間を比較的短くすることも可能になると考えられる。そのため、遺伝子回路を構成する分子を決定するとき、 タンパク質とRNAを適宜織り交ぜることで、転写調節から目的分子の細胞内の量を調節する時間をより広い幅でcontrolできるようになるかもしれない。</引用><br />
<br />
今回、私たちの用いるオシレーションのモデルは、<チューリングのアレ>です。このモデルにはactivatorとrepressorが必要です。それぞれの要素については以下に述べる。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Repressor===<br />
<p>We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. Some kinds of ncRNA work as a transcriptional repressor in vivo, for example, Gram-negative bacteria <i>Staphylococcus aureus</i> regulates a copy number of plasmid called pT181 in this mechanism.<sup>5</sup>The ncRNA in pT181 plasmid controls the fate of transcriptional elongation in response to an input by antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structures. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, RNA in attenuator region folds into an alternative structure which allows transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA and without other small molecules, many synthetic biologist constructed a variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). We chose this mechanism in gene repression. </p><br />
[[Image: 2013IGKUprojectRNArepressionMECHANISM.png]][[Image:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
<p>To ensure the function of antisense RNA and attenuator region, we will compare the amount of mRNA of GFP located in the downstream of an attenuator region in the presence and absence of antisense RNA.</p><br />
<br />
<p>転写の抑制を行うようなRNAの例として、我々は、伝令RNAに相補的に結合するncRNAによる転写制御を挙げる。これは、生体内でのRNAによるゲノム転写機構のひとつ、Gram-negative bacteria Staphylococcus aureusのpT181と呼ばれるplasmidなどのコピー数のregulationの機構である。RepressorとなるRNA (Antisense RNA)がある状態では、プロモーター下流のAttenuator locusがRho-independent terminator を形成することによりgenome coding部位の転写が抑制されるが、if the antisense RNA fails to bind, nascent RNA refolds into an alternative structure which prevents termination and promotes read-through (Novick, 1989) という仕組みを用いている。この機構は、他のリボスイッチと違いRNAのみで他の低分子化合物を用いていないため、合成生物学の新たな手法として、塩基置換などにより様々なタイプのものが作られている (Takahashi et al, 2013)。<br />
われわれはこれをRepressionの回路とした。AttenuatorとAntisenseの、Attenuator Regionより下流の遺伝子の転写を阻害する機能を確認するため、Attenuator antisense RNAの存在下と非存在下で、Attenuator Region下流のGFP遺伝子の発現量を比較した。 </p><br />
<p> In order to check the function of Attenuator and Antisense, we introduced Attenuator and Antisense into E.coli as experimental groups. We compared this E.coli with several controlled group in expression of GFP.</p><br />
<p>------const------</p><br />
Positive Control<br><br />
A-B: Pcon-RNAs(tetRaptamer, attenuator), Pcon-atte-GFP<br><br />
-antisenseを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、antisenseのもつ相補的配列が問題であることを確かめる。<br><br />
C: Pcon-atte-GFP<br><br />
Antisense非存在下においてはGFPの発現は抑制されないことを確認する。<br><br />
Experimental Group<br><br />
D: Pcon-antisense Pcon-atte-GFP<br><br />
Positive Control<br><br />
<br><br />
A-B. Pcon-RNAs(tetRaptamer, attenuator), Pcon-attenuator-GFP<br><br />
<p>In order to check the uniqueness of Antisense in repression, we introduced other RNAs into E.coli.</p><br />
C. Pcon-attenuator-GFP<br><br />
<p>This E.coli shows that if there is no Antisense, the expression of GFP is not repressed.</p><br />
Experimental Group<br><br />
D. Pcon-antisense Pcon-attenuator-GFP<br><br />
[[Image: IGKUprojectRNArepressionCONST2.png]]<br />
<p>------const------</p><br />
<p>---figcaption----</p><br />
<p> Antisenseが常時発現している大腸菌(figE)においてはAttenuator Regionの下流にあるGFPの転写が抑制され、Antisenseが存在しない大腸菌(figD)では抑制されていないことから、figEの大腸菌における転写抑制はAntisenseに起因することがわかる。figEの大腸菌でAntisenseをコードしていた部分を他の配列に置き換えた大腸菌(figA-C)におけるGFPの転写量はAntisenseを転写しない大腸菌(figD)に比べて遜色ないことから、figEの大腸菌での転写抑制はAtternatorに特異的なものであったことが導かれる。 </p><br />
<p> Compared with E.coli which didn’t express Antisense(figC), E.coli which always expresses Antisense(figD) was repressed in expression of GFP. This demonstrates that this repression was caused by expression of Antisense. Furthermore, because E.coli in which other structure of RNA was introduced(figA-B) expresses as much GFP as E.coli which didn’t expresses Antisense, we can say that this repression was peculiar to Antisense. </p><br />
<p>---figcaption----</p><br />
</div><br />
<div id="reportertab"><br />
<br />
===Activator===<br />
<p>We pick up tetR aptamer as an example of functional RNA which activates transcription. TetR aptamer specifically binds tet represser (tetR), which binds DNA specific site, repress transcription of downstream gene, and induce tetR conformational change and tetR reorientation.<sup>7</sup> That is, if in one cell, tetR is constantly expressed, gene located in the downstream of tet promoter is usually repressed and only when tetR aptamer is being expressed, it derepressed and transcribed. </p><br />
[[Image: 2013IGKUprojectRNAactivationMECHANISM.png]]<br />
<p>In our experiment, we check and measure tetR aptamer’s function in E.coli by comparing GFP fluorescence regulated by tetR promoter and GFP expression level by qRT-PCR in the following 4 cellular cases:1 TetR and tetR aptamer is constantly expressed. 2 Only tetR is constantly expressed and tetR aptamer is not induced.3 TetR and other functional RNA is constantly expressed. 4 TetR is not induced.</p><br />
<br />
<p>転写のアクチベーションを行うような機能性RNAの例として、我々はtetR aptamerを挙げる。これはtet repressorに特異的に結合するアプタマーであるが、DNAの特定領域に結合して転写を抑制しているtet repressorに結合してDNAから解離させる作用も持つ。つまり、常に一定量のtet repressorが発現し、存在しているような細胞内では、tetR aptamerが発現している間のみtet promotor以下の転写の抑制が解除、つまり活性化され、tetR aptamerが発現していず存在していない場合は、tetRの機能によって転写が抑制されるようになる。 tetR aptamerの働きを確認するため、tetRタンパク質とtetR aptamerを常時発現させた場合と、tetRタンパク質のみを常時発現させた場合、tetRとtetR アプタマー以外の構造をもつRNAを発現させた場合、tetRを発現させなかった場合とで、tetプロモーター下流に配置したGFP遺伝子を発現させその蛍光を見、qRT-PCRで発現量を比較しtetR aptamerの働きを確認した。(顕微鏡で蛍光度の差が確認できたときはqRT-PCRは補強扱いとし、確認できなかった場合はqRT-PCRのみを蛍光度の比較の尺度とする。) </p><br />
<p>In order to check the function of tetRaptamer, we introduced tetR and tetRaptamer as experimental group. We compared this E.coli with several controlled groups in expression of GFP.</p><br />
<p>-----コンストラクション------</p><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
-tetRを導入せず、Ptet-GFP単体のもの。tetRが存在しない場合にPtetがonになるということの確認。<br><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-TetR<br><br />
-tetRaptamerが存在しない場合。tetRがそのままで転写抑制をすることの確認。<br><br />
C-D. Ptet-GFP, Pcon-TetR, Pcon-RNAs(anti_attenuator, attenuator)<br><br />
-tetR aptamerを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、tetR aptamerのみが持つ構造と機能が問題であることを確かめる。<br><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-TetR, Pcon-tetRaptamer<br><br />
<br><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
<p>In order to check whether the GFP gene is expressed when there is no tetR protein, we introduced only Ptet-GFP.</p><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-tetR<br><br />
<p>Through this E.coli, it is confirmed that when tetR is expressed, tetR surpresses the expression of GFP.</p><br />
C-D. Ptet-GFP, Pcon-tetR, Pcon-RNAs(antisense, attenuator)<br><br />
<p>These kinds of E.coli shows that the surpression of the function of tetR is caused only by tetRaptamer.</p><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-tetR, Pcon-tetRaptamer<br><br />
[[Image: 2013IGKUprojectRNAactivatorCONST.png]]<br />
<p>-----コンストラクション------</p><br />
<p>-----fig Caption------</p><br />
<p> tetRを発現しない大腸菌(figA)の蛍光はtetRを発現する大腸菌(figB)のそれよりも強いことから、tetRはtetリプレッサーに結合して下流の転写を妨げることがわかる。tetRを発現し、tetRaptamerを転写しない大腸菌(figB)のGFP転写量に比べてtetRとtetRaptamerの両方を発現する大腸菌(figF)のGFP転写量が有意に大きいことから、tetRaptamerはtetRによる転写の抑制を解除する働きがあることが示唆される。tetRaptamerをコードしていた部分を他の配列に置き換えた大腸菌(figC-E)の蛍光はtetRとtetRaptamerの両方を発現する大腸菌(figF)よりも弱く、tetRのみを発現する大腸菌(figB)と同程度であることから、figFの大腸菌におけるtetRの機能の抑制はtetRaptamerに特有のものであることがわかる。</p><br />
<p> The fact that the fluorescence of E.coli which expressed tetR(figB) was weaker than E.coli which didn’t express tetR(figA) shows that tetR represses the expression of genes at the downstream of tet promotor. Since E.coli introduced tetR and tetRaptamer (figF) expressed more GFP than E.coli introduced only tetR(figB), we were confirmed that tetRaptamer cancels the effect of tetR {in some degree / almost completely}. Moreover, E.coli which was introduced other structure of RNA(figC-E) could express as much GFP as E.coli introduced tetR only(figB), which demonstrates that the surpression of the function of tetR protein is unique to tetRaptamer. </p><br />
<p>-----fig Caption------</p><br />
</div><br />
<div id="repressiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
【メモ:Assay、Result、Discussion】<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).<br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We performed RT-PCR to confirm tetR aptamer, antisense-spinach, spinach, and GFP<br />
--RT-PCRでの転写(機能)確認できた<br />
<br />
--Fusionの二次構造見た 影響しなさそうだった<br />
wetで事実作れる準備ができた<br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
===Achievement===<br />
我々は、このプロジェクトで以下のことを達成した。<br><br />
①<br><br />
②<br><br />
③<br><br />
④<br><br />
⑤<br><br />
⑥<br><br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-27T23:41:42Z<p>Niina: /* Conclusion */</p>
<hr />
<div>{{Kyoto/header}}<br />
<div id="kyoto-main"><br />
<html><br />
<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
</ul><br />
</html><br />
<div id="projectRNA"><br />
<html><br />
<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
</ul><br />
</html><br />
<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
<p>先のチューリングの例でみたとおり、場のスケールを多數の大腸菌で考えると菌体密度のようなfactorが影響してきてしまう。そのため、反応が複雑になってしまい、wetとdryの乖離を進める一因となっている。そこで、主要なファクターを考えやすくするために、場のスケールを一細胞で考えようと試みた。ここで扱うモデルは、よく研究されているオシレーションである。</p><br />
<p> As we state in Turing Model Project, if we try to make turing pattern with many cells of E.coli, we have to consider irregular factors such as density of cells. So, we have to simulate too complex circle to be practical. This complexity makes wet and dry unmatchable. So, then, we tried to compose turing pattern model on the inside of each E.coli’s cell. We adopted well-researched oscillation model. </p><br />
<br />
細胞内のオシレーションとしては、例えばシアノバクテリアのkai protein familyなどがある<reference>。Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。しかし、kaiのオシレーションを大腸菌で実現しようとすると。周期の時間がスケールが大腸菌の分裂速度よりも大幅に違う。よって、正確にモデリングすることが困難だろう。だから、短時間のモデルが考えやすいだろう。<br><br />
<p> In the natural world, for example, kai protein family oscillates in the cell of cyanobacteria. <reference> Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。However, because the cycle of kai protein family’s oscillation is much longer than E.coli’s cell cycle, it is difficult to create precise model of kai protein in E.coli. Hence, quicker oscillation is easy to simulate.</p><br />
<br />
さて、チューリングのモデルを大腸菌内で、かつ短い時間スケールで実現する際のfactorとして、私たちはRNAを提唱する。実際、転写調節因子としてのRNAの研究は始まっている<reference>。RNAを使う利点としては、次の2点が挙げられる。<br><br />
<p> Then, as quickly-oscillating factor in E.coli, we advocate functional RNA. In fact, research of RNA to regulate transcription have been undertaken. <reference> The merits of adopting functional RNA is following:</p><br />
<br />
<引用>4RNAは二次構造の予測や、RNA同士やDNAに対する特異的な結合を可能にするような設計を行うこともタンパク質に比較すると容易である。よって、遺伝子回路を製作するにあたって、回路を構成するRNA同士が塩基配列特異的な相互作用をするように設計すれば、数に限りがある既存のアクチベーターやリプレッサータンパク質を用いては不可能だったような、一細胞内で複数の独立した回路を共存させるということが可能になる。加えて、回路に直接関係しない任意の遺伝子の発現量をそれ同調させることも可能となる。<br />
<br />
Firstly, compared to protein, it is easier to predict the secondary structure of RNA, and to design the structure in order to bind to a specific RNA or DNA. Therefore, if we design the RNA which constructs the circuit to interact specifically to the base sequence, we can make some different circuits co-exist inside one cell. Moreover, since we can predict the structure we can link a post-transcriptional RNA reporter to the functional RNA to stop the conformational alternation, and realize an imaging of the RNA.<br />
<br />
5さらにRNAは転写後、機能するまでに翻訳の時間を要しないため、応答までの時間が短縮される。また、生体内での分解もタンパク質と比較して早いので、転写調節から応答までの時間を比較的短くすることも可能になると考えられる。そのため、遺伝子回路を構成する分子を決定するとき、 タンパク質とRNAを適宜織り交ぜることで、転写調節から目的分子の細胞内の量を調節する時間をより広い幅でcontrolできるようになるかもしれない。</引用><br />
<br />
今回、私たちの用いるオシレーションのモデルは、<チューリングのアレ>です。このモデルにはactivatorとrepressorが必要です。それぞれの要素については以下に述べる。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Repressor===<br />
<p>We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. Some kinds of ncRNA work as a transcriptional repressor in vivo, for example, Gram-negative bacteria <i>Staphylococcus aureus</i> regulates a copy number of plasmid called pT181 in this mechanism.<sup>5</sup>The ncRNA in pT181 plasmid controls the fate of transcriptional elongation in response to an input by antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structures. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, RNA in attenuator region folds into an alternative structure which allows transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA and without other small molecules, many synthetic biologist constructed a variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). We chose this mechanism in gene repression. </p><br />
[[Image: 2013IGKUprojectRNArepressionMECHANISM.png]][[Image:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
<p>To ensure the function of antisense RNA and attenuator region, we will compare the amount of mRNA of GFP located in the downstream of an attenuator region in the presence and absence of antisense RNA.</p><br />
<br />
<p>転写の抑制を行うようなRNAの例として、我々は、伝令RNAに相補的に結合するncRNAによる転写制御を挙げる。これは、生体内でのRNAによるゲノム転写機構のひとつ、Gram-negative bacteria Staphylococcus aureusのpT181と呼ばれるplasmidなどのコピー数のregulationの機構である。RepressorとなるRNA (Antisense RNA)がある状態では、プロモーター下流のAttenuator locusがRho-independent terminator を形成することによりgenome coding部位の転写が抑制されるが、if the antisense RNA fails to bind, nascent RNA refolds into an alternative structure which prevents termination and promotes read-through (Novick, 1989) という仕組みを用いている。この機構は、他のリボスイッチと違いRNAのみで他の低分子化合物を用いていないため、合成生物学の新たな手法として、塩基置換などにより様々なタイプのものが作られている (Takahashi et al, 2013)。<br />
われわれはこれをRepressionの回路とした。AttenuatorとAntisenseの、Attenuator Regionより下流の遺伝子の転写を阻害する機能を確認するため、Attenuator antisense RNAの存在下と非存在下で、Attenuator Region下流のGFP遺伝子の発現量を比較した。 </p><br />
<p> In order to check the function of Attenuator and Antisense, we introduced Attenuator and Antisense into E.coli as experimental groups. We compared this E.coli with several controlled group in expression of GFP.</p><br />
<p>------const------</p><br />
Positive Control<br><br />
A-B: Pcon-RNAs(tetRaptamer, attenuator), Pcon-atte-GFP<br><br />
-antisenseを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、antisenseのもつ相補的配列が問題であることを確かめる。<br><br />
C: Pcon-atte-GFP<br><br />
Antisense非存在下においてはGFPの発現は抑制されないことを確認する。<br><br />
Experimental Group<br><br />
D: Pcon-antisense Pcon-atte-GFP<br><br />
Positive Control<br><br />
<br><br />
A-B. Pcon-RNAs(tetRaptamer, attenuator), Pcon-attenuator-GFP<br><br />
<p>In order to check the uniqueness of Antisense in repression, we introduced other RNAs into E.coli.</p><br />
C. Pcon-attenuator-GFP<br><br />
<p>This E.coli shows that if there is no Antisense, the expression of GFP is not repressed.</p><br />
Experimental Group<br><br />
D. Pcon-antisense Pcon-attenuator-GFP<br><br />
[[Image: IGKUprojectRNArepressionCONST2.png]]<br />
<p>------const------</p><br />
<p>---figcaption----</p><br />
<p> Antisenseが常時発現している大腸菌(figE)においてはAttenuator Regionの下流にあるGFPの転写が抑制され、Antisenseが存在しない大腸菌(figD)では抑制されていないことから、figEの大腸菌における転写抑制はAntisenseに起因することがわかる。figEの大腸菌でAntisenseをコードしていた部分を他の配列に置き換えた大腸菌(figA-C)におけるGFPの転写量はAntisenseを転写しない大腸菌(figD)に比べて遜色ないことから、figEの大腸菌での転写抑制はAtternatorに特異的なものであったことが導かれる。 </p><br />
<p> Compared with E.coli which didn’t express Antisense(figC), E.coli which always expresses Antisense(figD) was repressed in expression of GFP. This demonstrates that this repression was caused by expression of Antisense. Furthermore, because E.coli in which other structure of RNA was introduced(figA-B) expresses as much GFP as E.coli which didn’t expresses Antisense, we can say that this repression was peculiar to Antisense. </p><br />
<p>---figcaption----</p><br />
</div><br />
<div id="reportertab"><br />
<br />
===Activator===<br />
<p>We pick up tetR aptamer as an example of functional RNA which activates transcription. TetR aptamer specifically binds tet represser (tetR), which binds DNA specific site, repress transcription of downstream gene, and induce tetR conformational change and tetR reorientation.<sup>7</sup> That is, if in one cell, tetR is constantly expressed, gene located in the downstream of tet promoter is usually repressed and only when tetR aptamer is being expressed, it derepressed and transcribed. </p><br />
[[Image: 2013IGKUprojectRNAactivationMECHANISM.png]]<br />
<p>In our experiment, we check and measure tetR aptamer’s function in E.coli by comparing GFP fluorescence regulated by tetR promoter and GFP expression level by qRT-PCR in the following 4 cellular cases:1 TetR and tetR aptamer is constantly expressed. 2 Only tetR is constantly expressed and tetR aptamer is not induced.3 TetR and other functional RNA is constantly expressed. 4 TetR is not induced.</p><br />
<br />
<p>転写のアクチベーションを行うような機能性RNAの例として、我々はtetR aptamerを挙げる。これはtet repressorに特異的に結合するアプタマーであるが、DNAの特定領域に結合して転写を抑制しているtet repressorに結合してDNAから解離させる作用も持つ。つまり、常に一定量のtet repressorが発現し、存在しているような細胞内では、tetR aptamerが発現している間のみtet promotor以下の転写の抑制が解除、つまり活性化され、tetR aptamerが発現していず存在していない場合は、tetRの機能によって転写が抑制されるようになる。 tetR aptamerの働きを確認するため、tetRタンパク質とtetR aptamerを常時発現させた場合と、tetRタンパク質のみを常時発現させた場合、tetRとtetR アプタマー以外の構造をもつRNAを発現させた場合、tetRを発現させなかった場合とで、tetプロモーター下流に配置したGFP遺伝子を発現させその蛍光を見、qRT-PCRで発現量を比較しtetR aptamerの働きを確認した。(顕微鏡で蛍光度の差が確認できたときはqRT-PCRは補強扱いとし、確認できなかった場合はqRT-PCRのみを蛍光度の比較の尺度とする。) </p><br />
<p>In order to check the function of tetRaptamer, we introduced tetR and tetRaptamer as experimental group. We compared this E.coli with several controlled groups in expression of GFP.</p><br />
<p>-----コンストラクション------</p><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
-tetRを導入せず、Ptet-GFP単体のもの。tetRが存在しない場合にPtetがonになるということの確認。<br><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-TetR<br><br />
-tetRaptamerが存在しない場合。tetRがそのままで転写抑制をすることの確認。<br><br />
C-D. Ptet-GFP, Pcon-TetR, Pcon-RNAs(anti_attenuator, attenuator)<br><br />
-tetR aptamerを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、tetR aptamerのみが持つ構造と機能が問題であることを確かめる。<br><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-TetR, Pcon-tetRaptamer<br><br />
<br><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
<p>In order to check whether the GFP gene is expressed when there is no tetR protein, we introduced only Ptet-GFP.</p><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-tetR<br><br />
<p>Through this E.coli, it is confirmed that when tetR is expressed, tetR surpresses the expression of GFP.</p><br />
C-D. Ptet-GFP, Pcon-tetR, Pcon-RNAs(antisense, attenuator)<br><br />
<p>These kinds of E.coli shows that the surpression of the function of tetR is caused only by tetRaptamer.</p><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-tetR, Pcon-tetRaptamer<br><br />
[[Image: 2013IGKUprojectRNAactivatorCONST.png]]<br />
<p>-----コンストラクション------</p><br />
<p>-----fig Caption------</p><br />
<p> tetRを発現しない大腸菌(figA)の蛍光はtetRを発現する大腸菌(figB)のそれよりも強いことから、tetRはtetリプレッサーに結合して下流の転写を妨げることがわかる。tetRを発現し、tetRaptamerを転写しない大腸菌(figB)のGFP転写量に比べてtetRとtetRaptamerの両方を発現する大腸菌(figF)のGFP転写量が有意に大きいことから、tetRaptamerはtetRによる転写の抑制を解除する働きがあることが示唆される。tetRaptamerをコードしていた部分を他の配列に置き換えた大腸菌(figC-E)の蛍光はtetRとtetRaptamerの両方を発現する大腸菌(figF)よりも弱く、tetRのみを発現する大腸菌(figB)と同程度であることから、figFの大腸菌におけるtetRの機能の抑制はtetRaptamerに特有のものであることがわかる。</p><br />
<p> The fact that the fluorescence of E.coli which expressed tetR(figB) was weaker than E.coli which didn’t express tetR(figA) shows that tetR represses the expression of genes at the downstream of tet promotor. Since E.coli introduced tetR and tetRaptamer (figF) expressed more GFP than E.coli introduced only tetR(figB), we were confirmed that tetRaptamer cancels the effect of tetR {in some degree / almost completely}. Moreover, E.coli which was introduced other structure of RNA(figC-E) could express as much GFP as E.coli introduced tetR only(figB), which demonstrates that the surpression of the function of tetR protein is unique to tetRaptamer. </p><br />
<p>-----fig Caption------</p><br />
</div><br />
<div id="repressiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
【メモ:Assay、Result、Discussion】<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
RT-PCR<br><br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
We performed RT-PCR to confirm tetR aptamer, antisense-spinach, spinach, and GFP<br />
--RT-PCRでの転写(機能)確認できた<br />
<br />
--Fusionの二次構造見た 影響しなさそうだった<br />
wetで事実作れる準備ができた<br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
===Achievement===<br />
我々は、このプロジェクトで以下のことを達成した。<br><br />
①<br><br />
②<br><br />
③<br><br />
④<br><br />
⑤<br><br />
⑥<br><br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
{{Kyoto/footer}}</div>Niinahttp://2013.igem.org/Team:Kyoto/projectRNATeam:Kyoto/projectRNA2013-09-27T23:36:55Z<p>Niina: /* Conclusion */</p>
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<div>{{Kyoto/header}}<br />
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<ul class="Kyoto-toptab"><br />
<li><a href="https://2013.igem.org/Kyoto:ProjectTuring"><img src="https://static.igem.org/mediawiki/2013/1/1f/Turingmodeltag.png"></a></li><br />
<li><a href="https://2013.igem.org/Kyoto:projectRNA"><img src="https://static.igem.org/mediawiki/2013/d/d8/RNAoscillatortag.png"></a></li><br />
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<div id="projectRNA"><br />
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<ul class="kyoto-tabs"><br />
<li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.png"></a></li><br><br />
<li><a href="#activationtab"><img src="https://static.igem.org/mediawiki/2013/f/fb/Activationtab.png"></a></li><br><br />
<li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.png"></a></li><br><br />
<li><a href="#fusiontab"><img src="https://static.igem.org/mediawiki/2013/d/dc/Fusiontab.png"></a></li><br><br />
<li><a href="#conctab"><img src="https://static.igem.org/mediawiki/2013/c/ce/Conclusiontab.png"></a></li><br><br />
<li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br><br />
<li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br><br />
<li><a href="#partslisttab"><img src="https://static.igem.org/mediawiki/2013/b/be/Partslisttab.png"></a></li><br><br />
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<div class="texts" style="margin-top: -9px;"><br />
=RNA Oscillator=<br />
<div id="introtab"><br />
==Introduction==<br />
===Motivation===<br />
<p>先のチューリングの例でみたとおり、場のスケールを多數の大腸菌で考えると菌体密度のようなfactorが影響してきてしまう。そのため、反応が複雑になってしまい、wetとdryの乖離を進める一因となっている。そこで、主要なファクターを考えやすくするために、場のスケールを一細胞で考えようと試みた。ここで扱うモデルは、よく研究されているオシレーションである。</p><br />
<p> As we state in Turing Model Project, if we try to make turing pattern with many cells of E.coli, we have to consider irregular factors such as density of cells. So, we have to simulate too complex circle to be practical. This complexity makes wet and dry unmatchable. So, then, we tried to compose turing pattern model on the inside of each E.coli’s cell. We adopted well-researched oscillation model. </p><br />
<br />
細胞内のオシレーションとしては、例えばシアノバクテリアのkai protein familyなどがある<reference>。Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。しかし、kaiのオシレーションを大腸菌で実現しようとすると。周期の時間がスケールが大腸菌の分裂速度よりも大幅に違う。よって、正確にモデリングすることが困難だろう。だから、短時間のモデルが考えやすいだろう。<br><br />
<p> In the natural world, for example, kai protein family oscillates in the cell of cyanobacteria. <reference> Kaiタンパクのオシレーションの機構は<ふにゃふにゃ>である。However, because the cycle of kai protein family’s oscillation is much longer than E.coli’s cell cycle, it is difficult to create precise model of kai protein in E.coli. Hence, quicker oscillation is easy to simulate.</p><br />
<br />
さて、チューリングのモデルを大腸菌内で、かつ短い時間スケールで実現する際のfactorとして、私たちはRNAを提唱する。実際、転写調節因子としてのRNAの研究は始まっている<reference>。RNAを使う利点としては、次の2点が挙げられる。<br><br />
<p> Then, as quickly-oscillating factor in E.coli, we advocate functional RNA. In fact, research of RNA to regulate transcription have been undertaken. <reference> The merits of adopting functional RNA is following:</p><br />
<br />
<引用>4RNAは二次構造の予測や、RNA同士やDNAに対する特異的な結合を可能にするような設計を行うこともタンパク質に比較すると容易である。よって、遺伝子回路を製作するにあたって、回路を構成するRNA同士が塩基配列特異的な相互作用をするように設計すれば、数に限りがある既存のアクチベーターやリプレッサータンパク質を用いては不可能だったような、一細胞内で複数の独立した回路を共存させるということが可能になる。加えて、回路に直接関係しない任意の遺伝子の発現量をそれ同調させることも可能となる。<br />
<br />
Firstly, compared to protein, it is easier to predict the secondary structure of RNA, and to design the structure in order to bind to a specific RNA or DNA. Therefore, if we design the RNA which constructs the circuit to interact specifically to the base sequence, we can make some different circuits co-exist inside one cell. Moreover, since we can predict the structure we can link a post-transcriptional RNA reporter to the functional RNA to stop the conformational alternation, and realize an imaging of the RNA.<br />
<br />
5さらにRNAは転写後、機能するまでに翻訳の時間を要しないため、応答までの時間が短縮される。また、生体内での分解もタンパク質と比較して早いので、転写調節から応答までの時間を比較的短くすることも可能になると考えられる。そのため、遺伝子回路を構成する分子を決定するとき、 タンパク質とRNAを適宜織り交ぜることで、転写調節から目的分子の細胞内の量を調節する時間をより広い幅でcontrolできるようになるかもしれない。</引用><br />
<br />
今回、私たちの用いるオシレーションのモデルは、<チューリングのアレ>です。このモデルにはactivatorとrepressorが必要です。それぞれの要素については以下に述べる。<br />
</div><br />
<div id="activationtab"><br />
<br />
===Repressor===<br />
<p>We took up non-coding RNA (ncRNA) complementarily binding mRNA as an example of functional RNA which repress transcription. Some kinds of ncRNA work as a transcriptional repressor in vivo, for example, Gram-negative bacteria <i>Staphylococcus aureus</i> regulates a copy number of plasmid called pT181 in this mechanism.<sup>5</sup>The ncRNA in pT181 plasmid controls the fate of transcriptional elongation in response to an input by antisense RNA. Attenuator region, which lies in 5' untranslated region of a transcript, folds into two different RNA structures. By an interaction with complementary antisense RNA, attenuator region forms Rho-independent terminator and the transcription of the downstream is stopped. Without antisense RNA, RNA in attenuator region folds into an alternative structure which allows transcription of the downstream (Novick, 1989). The uniqueness of this mechanism is that it is constructed with only RNA and without other small molecules, many synthetic biologist constructed a variant of it by means of nucleotide substitution etc. (Takahashi et al, 2013). We chose this mechanism in gene repression. </p><br />
[[Image: 2013IGKUprojectRNArepressionMECHANISM.png]][[Image:2013IGKUprojectRNArepressionMECHANISM2.png]]<br />
<p>To ensure the function of antisense RNA and attenuator region, we will compare the amount of mRNA of GFP located in the downstream of an attenuator region in the presence and absence of antisense RNA.</p><br />
<br />
<p>転写の抑制を行うようなRNAの例として、我々は、伝令RNAに相補的に結合するncRNAによる転写制御を挙げる。これは、生体内でのRNAによるゲノム転写機構のひとつ、Gram-negative bacteria Staphylococcus aureusのpT181と呼ばれるplasmidなどのコピー数のregulationの機構である。RepressorとなるRNA (Antisense RNA)がある状態では、プロモーター下流のAttenuator locusがRho-independent terminator を形成することによりgenome coding部位の転写が抑制されるが、if the antisense RNA fails to bind, nascent RNA refolds into an alternative structure which prevents termination and promotes read-through (Novick, 1989) という仕組みを用いている。この機構は、他のリボスイッチと違いRNAのみで他の低分子化合物を用いていないため、合成生物学の新たな手法として、塩基置換などにより様々なタイプのものが作られている (Takahashi et al, 2013)。<br />
われわれはこれをRepressionの回路とした。AttenuatorとAntisenseの、Attenuator Regionより下流の遺伝子の転写を阻害する機能を確認するため、Attenuator antisense RNAの存在下と非存在下で、Attenuator Region下流のGFP遺伝子の発現量を比較した。 </p><br />
<p> In order to check the function of Attenuator and Antisense, we introduced Attenuator and Antisense into E.coli as experimental groups. We compared this E.coli with several controlled group in expression of GFP.</p><br />
<p>------const------</p><br />
Positive Control<br><br />
A-B: Pcon-RNAs(tetRaptamer, attenuator), Pcon-atte-GFP<br><br />
-antisenseを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、antisenseのもつ相補的配列が問題であることを確かめる。<br><br />
C: Pcon-atte-GFP<br><br />
Antisense非存在下においてはGFPの発現は抑制されないことを確認する。<br><br />
Experimental Group<br><br />
D: Pcon-antisense Pcon-atte-GFP<br><br />
Positive Control<br><br />
<br><br />
A-B. Pcon-RNAs(tetRaptamer, attenuator), Pcon-attenuator-GFP<br><br />
<p>In order to check the uniqueness of Antisense in repression, we introduced other RNAs into E.coli.</p><br />
C. Pcon-attenuator-GFP<br><br />
<p>This E.coli shows that if there is no Antisense, the expression of GFP is not repressed.</p><br />
Experimental Group<br><br />
D. Pcon-antisense Pcon-attenuator-GFP<br><br />
[[Image: IGKUprojectRNArepressionCONST2.png]]<br />
<p>------const------</p><br />
<p>---figcaption----</p><br />
<p> Antisenseが常時発現している大腸菌(figE)においてはAttenuator Regionの下流にあるGFPの転写が抑制され、Antisenseが存在しない大腸菌(figD)では抑制されていないことから、figEの大腸菌における転写抑制はAntisenseに起因することがわかる。figEの大腸菌でAntisenseをコードしていた部分を他の配列に置き換えた大腸菌(figA-C)におけるGFPの転写量はAntisenseを転写しない大腸菌(figD)に比べて遜色ないことから、figEの大腸菌での転写抑制はAtternatorに特異的なものであったことが導かれる。 </p><br />
<p> Compared with E.coli which didn’t express Antisense(figC), E.coli which always expresses Antisense(figD) was repressed in expression of GFP. This demonstrates that this repression was caused by expression of Antisense. Furthermore, because E.coli in which other structure of RNA was introduced(figA-B) expresses as much GFP as E.coli which didn’t expresses Antisense, we can say that this repression was peculiar to Antisense. </p><br />
<p>---figcaption----</p><br />
</div><br />
<div id="reportertab"><br />
<br />
===Activator===<br />
<p>We pick up tetR aptamer as an example of functional RNA which activates transcription. TetR aptamer specifically binds tet represser (tetR), which binds DNA specific site, repress transcription of downstream gene, and induce tetR conformational change and tetR reorientation.<sup>7</sup> That is, if in one cell, tetR is constantly expressed, gene located in the downstream of tet promoter is usually repressed and only when tetR aptamer is being expressed, it derepressed and transcribed. </p><br />
[[Image: 2013IGKUprojectRNAactivationMECHANISM.png]]<br />
<p>In our experiment, we check and measure tetR aptamer’s function in E.coli by comparing GFP fluorescence regulated by tetR promoter and GFP expression level by qRT-PCR in the following 4 cellular cases:1 TetR and tetR aptamer is constantly expressed. 2 Only tetR is constantly expressed and tetR aptamer is not induced.3 TetR and other functional RNA is constantly expressed. 4 TetR is not induced.</p><br />
<br />
<p>転写のアクチベーションを行うような機能性RNAの例として、我々はtetR aptamerを挙げる。これはtet repressorに特異的に結合するアプタマーであるが、DNAの特定領域に結合して転写を抑制しているtet repressorに結合してDNAから解離させる作用も持つ。つまり、常に一定量のtet repressorが発現し、存在しているような細胞内では、tetR aptamerが発現している間のみtet promotor以下の転写の抑制が解除、つまり活性化され、tetR aptamerが発現していず存在していない場合は、tetRの機能によって転写が抑制されるようになる。 tetR aptamerの働きを確認するため、tetRタンパク質とtetR aptamerを常時発現させた場合と、tetRタンパク質のみを常時発現させた場合、tetRとtetR アプタマー以外の構造をもつRNAを発現させた場合、tetRを発現させなかった場合とで、tetプロモーター下流に配置したGFP遺伝子を発現させその蛍光を見、qRT-PCRで発現量を比較しtetR aptamerの働きを確認した。(顕微鏡で蛍光度の差が確認できたときはqRT-PCRは補強扱いとし、確認できなかった場合はqRT-PCRのみを蛍光度の比較の尺度とする。) </p><br />
<p>In order to check the function of tetRaptamer, we introduced tetR and tetRaptamer as experimental group. We compared this E.coli with several controlled groups in expression of GFP.</p><br />
<p>-----コンストラクション------</p><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
-tetRを導入せず、Ptet-GFP単体のもの。tetRが存在しない場合にPtetがonになるということの確認。<br><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-TetR<br><br />
-tetRaptamerが存在しない場合。tetRがそのままで転写抑制をすることの確認。<br><br />
C-D. Ptet-GFP, Pcon-TetR, Pcon-RNAs(anti_attenuator, attenuator)<br><br />
-tetR aptamerを他のRNAで置き換えたもの。これによってRNAであることが問題なのでなく、tetR aptamerのみが持つ構造と機能が問題であることを確かめる。<br><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-TetR, Pcon-tetRaptamer<br><br />
<br><br />
Positive Control<br><br />
A. Ptet-GFP<br><br />
<p>In order to check whether the GFP gene is expressed when there is no tetR protein, we introduced only Ptet-GFP.</p><br />
Negative Control<br><br />
B. Ptet-GFP, Pcon-tetR<br><br />
<p>Through this E.coli, it is confirmed that when tetR is expressed, tetR surpresses the expression of GFP.</p><br />
C-D. Ptet-GFP, Pcon-tetR, Pcon-RNAs(antisense, attenuator)<br><br />
<p>These kinds of E.coli shows that the surpression of the function of tetR is caused only by tetRaptamer.</p><br />
Experimental Group<br><br />
E. Ptet-GFP, Pcon-tetR, Pcon-tetRaptamer<br><br />
[[Image: 2013IGKUprojectRNAactivatorCONST.png]]<br />
<p>-----コンストラクション------</p><br />
<p>-----fig Caption------</p><br />
<p> tetRを発現しない大腸菌(figA)の蛍光はtetRを発現する大腸菌(figB)のそれよりも強いことから、tetRはtetリプレッサーに結合して下流の転写を妨げることがわかる。tetRを発現し、tetRaptamerを転写しない大腸菌(figB)のGFP転写量に比べてtetRとtetRaptamerの両方を発現する大腸菌(figF)のGFP転写量が有意に大きいことから、tetRaptamerはtetRによる転写の抑制を解除する働きがあることが示唆される。tetRaptamerをコードしていた部分を他の配列に置き換えた大腸菌(figC-E)の蛍光はtetRとtetRaptamerの両方を発現する大腸菌(figF)よりも弱く、tetRのみを発現する大腸菌(figB)と同程度であることから、figFの大腸菌におけるtetRの機能の抑制はtetRaptamerに特有のものであることがわかる。</p><br />
<p> The fact that the fluorescence of E.coli which expressed tetR(figB) was weaker than E.coli which didn’t express tetR(figA) shows that tetR represses the expression of genes at the downstream of tet promotor. Since E.coli introduced tetR and tetRaptamer (figF) expressed more GFP than E.coli introduced only tetR(figB), we were confirmed that tetRaptamer cancels the effect of tetR {in some degree / almost completely}. Moreover, E.coli which was introduced other structure of RNA(figC-E) could express as much GFP as E.coli introduced tetR only(figB), which demonstrates that the surpression of the function of tetR protein is unique to tetRaptamer. </p><br />
<p>-----fig Caption------</p><br />
</div><br />
<div id="repressiontab"><br />
<br />
===Reporter===<br />
我々は、RNAでできたレポーターとなりうる分子として、Spinachを挙げる。これはJeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, によって設計されたアプタマーの一種で、GFPを模倣している。SpinachはGFPの蛍光部位によく似た合成物であるDMHBIに特異的に結合するアプタマーから設計された。GFPのfluorophoreはdenatured GFPでは蛍光を示すことがなく、分子の奥に折りたたまれて初めて蛍光を発するようになる。DMHBIもこれと似た性質を持っており、単体ではほぼ蛍光を示すことはなく、GFPの構造の持つ機能を真似たSpinachの高次構造の奥に取り込まれて初めて蛍光するようになる。そのため、サンプルにDMHBIを加えた後に蛍光を確認すると、サンプル内にSpinachが存在するかどうかがわかる。もし存在すればSpinachはDMHBIと結合して蛍光を発するだろうし、存在しなければ蛍光は発しえない。Spinachを用いることで、RNAを直接イメージングできる他、安定なタンパク質では確認できない、大きく変化するRNAの発現量を正確に反映することが出来る。<br><br />
<br />
Spinach is an example of a reporter molecular, which is a kind of aptamer designed by Jeremy S. Paige, Karen Y. Wu and Samie R. Jaffrey imitating GFP. It is designed from aptamer combining with the complex--DMHBI which is considerably similar to the fluorescence site of GFP. The fluorescence of GFP show as long as the molecular is folded in the back of the molecule, instead in denatured GFP. DMHBI also shows almost the same property--the simple substance produces little fluorescence, compared with when captured by higher-order constructions. That is to say, if fluorescence is conformed after DMHBI is added, it is manifest that Spinach exists. If Spinach exists, it combines with DMHBI to produce fluorescence, vice versa. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the expression level which vary intensely, which can’t be confirmed via stable protein. <br />
<br><br><br />
We suggest Spinach as a molecule which can be a reporter made of RNA. It is an aptamer designed by Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey and imitates GFP. Spinach was designed from the aptamer which binds specifically to DMHBI, which is similar to fluorophore of GFP.<br />
<br />
【メモ:Assay、Result、Discussion】<br />
</div><br />
<div id="fusiontab"><br />
<br />
===Fusion===<br />
<p>これらを使って遺伝子回路を組み立てるとき、複数のmoduleを同じ機能要素に組みこまなければならないときも十分あり得る。例えば転写抑制の様子をレポートするとき、異なる因子で促進と抑制を行うような系を作るときである。このとき、複数のモジュールを連結したことによる相互作用や立体構造の問題により機能が阻害される可能性がある。タンパク質ではその問題を予測するのは難しいが、RNAであれば配列情報から比較的簡単に二次構造を予測することができ、これらの問題を回避出来る。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, をそれぞれつなぎあわせ、二次構造を予測し、実際に働いていることを確認した。tetRタンパク質存在下でtetR aptamerとAttenuator antisense RNAを組み合わせたRNAがPtetプロモーター下流のGFPの転写量を増加させるかを確認した。<br />
並びにAttenuator antisense RNAとSpinarchを連結したRNAを発現させ、Attenuator Region下流のGFP遺伝子の発現量が減少していることとSpinarchがDFHBI存在下で蛍光するかどうかを確認した。<br />
<br />
Intending to check the process of transcriptional repression system and the system which promotes and represses processes of transcription by using different factors, we have to join some modules into a single RNA strand.<br />
When we combine plural modules, the function of the modules may be inhibited by interactions and steric structures between each other. in the case of RNA,it is easier to predict and avoid the steric problems than that of proteins because we can predict the secondary structure of RNA from its primary structure. We combined tetR aptamer and Antisense-Attenuator RNA, whose functions are confirmed, and predicted secondary structures, as a result it actually worked. We also observed tetR aptamer-Attenuator antisense fusion RNA increased expression level of downstream GFP of tet promoter in the presence of tetR proteins.<br />
<br><br />
</p><br />
<p>-----const-------</p><br />
experimental group<br><br />
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
positive control<br><br />
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br><br />
-Fusionする前とのtetRaptamerの働きの比較<br><br />
negative control<br><br />
c. Ptet-GFP-DT Pcon-tetR-DT<br><br />
<br><br />
Positive Control<br><br />
A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
<p>Through this E.coli, we can confirm that separated tetRaptamer restricts the function of tetR protein.</p><br />
Negative Control<br><br />
B. Ptet-GFP Pcon-tetR<br><br />
<p>This E.coli shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p><br />
Experimental Group<br><br />
C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br><br />
[[Image: 2013IGKUprojectRNAfusionCONST2.png]]<br />
<p>------const-------</p><br />
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown below, the structure of tetR aptamer is not affected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not affected by the existence of attenuator stem loop.</p> <br />
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p><br />
<p> tetRが常時発現されている状態では、AttenuatorとtetRaptamerを連結したRNAを転写する大腸菌(figC)のGFP発現量は、tetRaptamerを転写しない大腸菌(figB)よりも多く、ほかのRNAと連結していないtetRaptamerを転写する大腸菌(figA)と比較して{ほぼ同等 or 小さい}であることから、AttenuatorとtetRaptamerを連結すると、tetRaptamerは{全く干渉せずに機能する or 効果は下がるが機能する}ことがわかる。 </p><br />
<p> If tetR is expressed, E.coli in which the united RNA(figC) was introduced expresses more GFP than E.coli which didn’t have the tetRaptamer sequence(figB). By comparing E.coli expressing independent tetRaptamer(figA) and E.coli expressing the united RNA(figC), it is convinced that tetRaptamer next door to Attenuator { works as well as independent one / works more weakly than a independent one. However, it certainly works.} </p><br />
</div><br />
<div id="conctab"><br />
<br />
==Experiment==<br />
(・Construction)<br />
・転写確認<br />
・構造予測<br />
</div><br />
<br />
==Result==<br />
(・コンスト泳動結果)<br><br />
===RT-PCR===<br />
RT-PCR<br><br />
[[File:ElectrophoresisRT]]<br />
<br />
===Structure Prediction===<br />
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]<br />
[[File:antisense_spinach.png]]with Centroid Fold*<br />
<br><br />
<br />
==Conclusion==<br />
--RT-PCRでの転写(機能)確認できた<br />
--Fusionの二次構造見た 影響しなさそうだった<br />
wetで事実作れる準備ができた<br />
<div id="futuretab"><br />
<br />
==future work==<br />
・定数探し<br />
・シュミレーションする<br />
・Attenuator-tetR アプタマーのコンストラクション<br />
・機能確認<br />
・実際にwetでオシレーションができることをみる<br />
・wetとdryを比較する<br />
<br />
==文章のたまり場==<br />
===旧conclusion===<br />
In this project we confirmed the function of activator (tetR / tetR aptamer) and repressor (Attenuator region / Antisense). Moreover, we predicted the secondary structure of linked RNA (Antisense RNA-tetR aptamer) to check the influence of linkage to the structure, and finally we confirmed that actually the function of tetR aptamer do not lost. Our outcome of this project will directly connects to the progress of synthetic biology, especially constructing gene circuits. These two types of functional RNA will play important role when we regulate gene expression in peculiar gene cycle. For example, we may regulate a gene circuit which contains rapid gene transcriptional cascades by these RNA modules.<br />
<br />
==Future Work==<br />
<p> To show this possibility, we designed a gene circuit which uses these RNA module. This circuit produces transcriptional oscillation. Oscillation circuits are important and essential gene circuits in many organisms and always be in the center of synthetic biology, therefore it is suitable for the cutting edge of new type of gene regulation. </p><br />
<p> When it comes to oscillation, we have to have a module which acts as reporter to show the changing amount of post-transcriptional RNA. Usually, protein reporters such as GFP are used for this purpose. However, in this circuit protein reporters may not be able to be used, because of the length of the period of the oscillation. Because RNA’s degradation is so fast and RNA do not need to be translated or folded like protein, the period of oscillation should be too short. According to XX who constructed similar gene circuit using RNA modules, this kinds of circuit produces 10 minutes cycle reaction. This means protein degradation is too slow (takes XX hours even with the degradation tag) [要出典] to image this RNA oscillation.</p><br />
<p> To solve this problem, we will suggest a new RNA module, which called spinach. This is a kind of aptamer, which is designed by Jeremy S. Paige, Karen Y. Wu, and Samie R. Jaffrey.<sup>20</sup> They imitated the structure of GFP in this project. The designing of Spinach is changing the structure of an aptamer which specifically combines with DFHBI, which has similar structure to fluorophore of GFP. Denatured GFP doesn’t have fluorescence. Only if GFP is folded correctly, the fluorophore of GFP, which is in inner area, emits fluorescence. Therefore, we can confirm whether there is Spinarch in a sample by adding DFHBI. If the sample contains Spinarch, the sample will emit fluorescent. Vice versa. Spinach may degrade first enough for the oscillation, therefore we propose this for reporter of this oscillation.</p><br />
<p>The circuit of oscillation which uses spinach and the two RNA module is like below. This describes mechanism of producing oscillation.</p><br />
<br />
<html><br />
<center><br />
<iframe id="kyoto_prezi" src="http://prezi.com/embed/eaubz-cct4kd/?bgcolor=ffffff&amp;lock_to_path=0&amp;autoplay=0&amp;autohide_ctrls=0&amp;features=undefined&amp;disabled_features=undefined" width="550" height="400" frameBorder="0"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/><br />
</iframe><br />
</center><br />
</html><br/><br />
<br />
<p>この回路がオシレーションを形成する仕組みは、以下のようになっている。初期条件として、Constitutive Promoterにより合成されたTetRにより、Ptetはrepressされている。 オシレーションの開始はPtet下流のPlacがIPTGにより誘導されることである。これによってRNA-Actが合成開始され、その中のtetR aptamer配列がPtetをactivateする。 ActivateされたPtetはさらにRNA-Actを合成し、ここでポジティブ・フィードバックがかかることでRNA-Act, RNA-Repともにその量を増やす。すると、RNA-Repの配列内のSpinachにより緑色蛍光が確認される。 RNA-Repの量が十分に増えると、そのAttenuator antisenseの部位がRNA-ActのAttenuator locusに結合し、RNA-Actの転写量を減少させる。 するとTetR-AptamerによるActivationが小さくなることで、RNA-Act, RNA-Repの量が減少する。すると、Spinachによる蛍光は減衰する。 RNA-Repの量が十分に減少すると、Attenuator antisenseによる転写抑制が解かれ、再びRNA-Actの転写量が増えることとなる。これが繰り返されることで、オシレーションを作り上げている。この回路からは、RNAならではの分解・生成が速い性質によって、10分周期程度の短いSpinach蛍光のオシレーションを生むことが出来ると予測できる。</p><br />
<p> This circuit oscillates in the following way: First, tet promoter is repressed by tetR at the downstream of constitutive promotor. Then, the oscillator is turned on by IPTG. IPTG activates Plac and tetRaptamer, Spinarch, and Antisense at the downstream of Ptet which are transcribed. Because tetRaptamer activates tet promotor, positive feedback occurs and more and more tetRaptamer, Spinarch, and Antisense are accumulated. Then, this circuit gets fluorescence. After Antisense is accumulated to some extent, tetRaptamer, at the downstream of Atteruator region, is repressed. Then, because new tetRaptamer is not created, the amount of tetRaptamer decreases quickly. So, tet promotor is repressed by tetR protein and the amount of Antisense and Spinarch falls, too. Then, this circuit loses fluorescence. After the amount of Antisense decreases sufficiently, this circuit recovers first condition. Through this cycle, this circuit acts as an oscillator. Since RNA is generated and resolved quickly, this circuit should oscillate as quick as about in 10 minutes' cycle. </p><br />
<br><br />
&#9757;&#9757;たぶん時制がめちゃくちゃですごめんなさい(´._.`)<br />
</div><br />
<div id="achievetab"><br />
<br />
===Achievement===<br />
我々は、このプロジェクトで以下のことを達成した。<br><br />
①<br><br />
②<br><br />
③<br><br />
④<br><br />
⑤<br><br />
⑥<br><br />
</div><br />
<div id="partslisttab"><br />
<br />
== Parts List ==<br />
<groupparts>iGEM013 Kyoto</groupparts><br />
</div><br />
<div id="referencetab"><br />
<br />
== Reference ==<br />
[7(仮)][http://www.ncbi.nlm.nih.gov/pubmed/19246008 Anke Hunsicker et al.(2009)"An RNA aptamer that induces transcription"Chem Biol,16(2),173-180]<br><br />
[20(仮)][http://www.sciencemag.org/content/333/6042/642.abstract Jeremy S. Paige et al.(2011)"RNA Mimics of Green Fluorescent Protein"Science Vol. 333 no. 6042 pp. 642-646]<br><br />
[5(仮)][http://www.ncbi.nlm.nih.gov/pubmed/23761434 Melissa K. Takahashi and Julius B. Lucks.(2013)"A modular strategy for engineering orthogonal chimeric RNA transcription regulators"Nucleic Acids Research 41(15),7577-88]<br><br />
[http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br />
</div><br />
</div><br />
</div><br />
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