Team:Kyoto/projectRNA

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<div id="projectRNA">
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  <li><a href="#introtab"><img src="https://static.igem.org/mediawiki/2013/b/be/Introductiontab.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>
 
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  <li><a href="#repressiontab"><img src="https://static.igem.org/mediawiki/2013/1/1e/RNArepressiontab.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>
 
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  <li><a href="#futuretab"><img src="https://static.igem.org/mediawiki/2013/7/73/RNAFutureworktab.png"></a></li><br>
 
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  <li><a href="#achievetab"><img src="https://static.igem.org/mediawiki/2013/f/fe/RNAtabAchievement.png"></a></li><br>
 
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<div class="texts" style="margin-top: -9px;">
<div class="texts" style="margin-top: -9px;">
=RNA Oscillator=
=RNA Oscillator=
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==Introduction==
==Introduction==
===Motivation===
===Motivation===
-
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.  
+
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.  
-
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.
+
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.[1] 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.
</div>
</div>
<div id="activationtab">
<div id="activationtab">
===Oscillation===
===Oscillation===
-
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.
+
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.
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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 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.
+
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.
-
 
+
[[File:Kyoto_RNA_Prezi.png]]
</div>
</div>
<div id="reportertab">
<div id="reportertab">
===Repressor===
===Repressor===
-
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.  
+
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)[5]. 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].
 +
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
 +
[[File:2013IGKUprojectRNArepressionMECHANISM.png]]
 +
[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]
</div>
</div>
<div id="repressiontab">
<div id="repressiontab">
===Activator===
===Activator===
-
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. ).
+
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.
 +
[[File:No-binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]
</div>
</div>
<div id="fusiontab">
<div id="fusiontab">
===Reporter===
===Reporter===
-
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>
+
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>
-
 
+
-
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>
+
We strongly suggest Spinach aptamer as a reporter of RNA.
We strongly suggest Spinach aptamer as a reporter of RNA.
 +
[[File:SPINACHの説明.png]]
===Fusion===
===Fusion===
-
<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。
+
<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.
-
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。
+
We estimated the RNA structure to check whether or not unindicatd duplex is formed by open tool.
-
 
+
-
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.
+
-
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>
+
</p>
</p>
-
<p>-----const-------</p>
 
-
experimental group<br>
 
-
a. Pcon-atte-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br>
 
-
positive control<br>
 
-
b. Pcon-tetRaptamer-DT Ptet-GFP-DT Pcon-tetR-DT<br>
 
-
-Fusionする前とのtetRaptamerの働きの比較<br>
 
-
negative control<br>
 
-
c. Ptet-GFP-DT Pcon-tetR-DT<br>
 
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<br>
 
-
Positive Control<br>
 
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A. Pcon-tetRaptamer Ptet-GFP Pcon-tetR<br>
 
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<p>Through this ''E.coli'', we can confirm that separated tetRaptamer restricts the function of tetR protein.</p>
 
-
Negative Control<br>
 
-
B. Ptet-GFP Pcon-tetR<br>
 
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<p>This ''E.coli'' shows that tetR protein represses the expression of genes at the downstream of tet promotor.</p>
 
-
Experimental Group<br>
 
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C. Pcon-atte-tetRaptamer Ptet-GFP Pcon-tetR<br>
 
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[[Image: 2013IGKUprojectRNAfusionCONST2.png]]
 
-
<p>------const-------</p>
 
-
<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>
 
-
[[Image: 2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]
 
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<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisense</p>
 
-
<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>
 
-
<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>
 
</div>
</div>
<div id="conctab">
<div id="conctab">
==Experiment==
==Experiment==
-
After we construct functional RNA generator, we checked whether transcription of the RNA parts. To confirm this, we performed RT-PCR.<br>
+
After we constructed functional RNA generator, we checked the transcription of the RNA parts. To confirm this, we performed RT-PCR.<br>
samples are following:<br>
samples are following:<br>
Negative control<br>
Negative control<br>
-
*Spinach-DT
+
*Non-promoter: Spinach-DT
Experimental group<br>
Experimental group<br>
[[File:唯一のexperiment.png]]<br>
[[File:唯一のexperiment.png]]<br>
-
We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]
+
We also checked whether fusion RNA we designed functions or not considering secondary structure with Centroid Fold[6]
</div>
</div>
==Result==
==Result==
===RT-PCR===
===RT-PCR===
-
We performed RT-PCR to confirm transcription of tetR aptamer, antisense-spinach, spinach, and GFP(GFP generator).
+
We performed RT-PCR to confirm transcription of TetR aptamer(left) and Spinach(center).<br>
-
[[File:ElectrophoresisRT]]
+
[[File:ElectrophoresisRT1.png]]
 +
[[File:ElectrophoresisRT2.png]]
 +
 
===Structure Prediction===
===Structure Prediction===
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]
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==Conclusion==
==Conclusion==
-
We confirmed transcription of tetR aptamer, antisense-spinach, spinach, and GFP by using RT-PCR method.<br>
+
We confirmed the transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP by using RT-PCR method.<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>
+
We predicted secondary structure of fusion RNA: atenuator-TetRaptamer and antisence-Spinach with centroid fold. It seems to be the expected structure and to function as expected.<br>
-
We got ready for construction oscilator circuit in wet lab.<br>
+
We got ready for the construction of the oscilator circuit in wet lab.<br>
<div id="futuretab">
<div id="futuretab">
==Future work==
==Future work==
-
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>
+
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>
-
After that, we will substitute the values for oscilation model and try to solve simulate. And we will continue assaying of our parts.<br>
+
1. qualitative assay TatR aptamer<br>
 +
To confirm the act of TetR aptamer inducing Ptet ,we are constructing IPTG-inducble TetR aptamer to express GFP. As negative controls, we use RNA with antisense, attenuator, Spinach, no-RNA and attenuator-TetR aptamer. As positive controls, GFP is constitutively expressed.<br>
 +
3, qualitatively Spinach assay (visual recognition & fluorescence microscopes)<br>
 +
We will check that DFHBI fluorescence on a plate with Spinach.<br>
 +
We will 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>
 +
After that, we will substitute the values for oscilation model and try to solve simulate. Moreover we will continue assaying of our parts.<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>
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>
-
Finaly, we compair results of wet lab and dry lab and discuss a point in common/difference between the results.
+
Finaly, we compare results of wet lab and dry lab and discuss a point in common/difference between the results.
</div>
</div>
<div id="achievetab">
<div id="achievetab">
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== Reference ==
== Reference ==
-
[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>
+
[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>
[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>
[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>
[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>
[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>
[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>
[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>
-
[5][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]
+
[5][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>
 +
[6][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br>
 +
 
</div>
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{{Kyoto/import|pagename={{PAGENAME}}}}
 
{{Kyoto/footer}}
{{Kyoto/footer}}

Latest revision as of 12:45, 10 October 2013

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Contents

RNA Oscillator

Introduction

Motivation

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. 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.[1] 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.

Oscillation

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.

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. Kyoto RNA Prezi.png

Repressor

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)[5]. 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]. 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 2013IGKUprojectRNArepressionMECHANISM.png 2013IGKUprojectRNArepressionMECHANISM2.png

Activator

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. No-binding-of-tetR-aptamer.pngBinding-of-tetR-aptamer.png

Reporter

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.
We strongly suggest Spinach aptamer as a reporter of RNA. SPINACHの説明.png

Fusion

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. We estimated the RNA structure to check whether or not unindicatd duplex is formed by open tool.

Experiment

After we constructed functional RNA generator, we checked the transcription of the RNA parts. To confirm this, we performed RT-PCR.
samples are following:
Negative control

  • Non-promoter: Spinach-DT

Experimental group
唯一のexperiment.png
We also checked whether fusion RNA we designed functions or not considering secondary structure with Centroid Fold[6]

Result

RT-PCR

We performed RT-PCR to confirm transcription of TetR aptamer(left) and Spinach(center).
ElectrophoresisRT1.png ElectrophoresisRT2.png

Structure Prediction

2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png Antisense spinach.pngwith Centroid Fold*

Conclusion

We confirmed the transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP by using RT-PCR method.
We predicted secondary structure of fusion RNA: atenuator-TetRaptamer and antisence-Spinach with centroid fold. It seems to be the expected structure and to function as expected.
We got ready for the construction of the oscilator circuit in wet lab.

Future work

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.
1. qualitative assay TatR aptamer
To confirm the act of TetR aptamer inducing Ptet ,we are constructing IPTG-inducble TetR aptamer to express GFP. As negative controls, we use RNA with antisense, attenuator, Spinach, no-RNA and attenuator-TetR aptamer. As positive controls, GFP is constitutively expressed.
3, qualitatively Spinach assay (visual recognition & fluorescence microscopes)
We will check that DFHBI fluorescence on a plate with Spinach.
We will 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.
After that, we will substitute the values for oscilation model and try to solve simulate. Moreover we will continue assaying of our parts.
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 this page
Finaly, we compare results of wet lab and dry lab and discuss a point in common/difference between the results.

Parts List

<groupparts>iGEM013 Kyoto</groupparts>

Reference

[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]
[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]
[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]
[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]
[5][http://www.ncbi.nlm.nih.gov/pubmed/2478296 Novick RP et al. (1999) "pT181 Plasmid Replication Is Regulated by a Countertranscript-Driven Transcriptional Attenuator"]
[6][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]

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