Team:Kyoto/projectRNA

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= RNA Oscillator=
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<ul class="Kyoto-toptab">
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== motivation ==
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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>
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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>
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</ul>
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</html>
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<div id="projectRNA">
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<div class="texts" style="margin-top: -9px;">
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=RNA Oscillator=
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<div id="introtab">
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==Introduction==
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===Motivation===
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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.
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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.
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</div>
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<div id="activationtab">
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　人の手で様々な遺伝子を組み合わせて生体の複雑な遺伝子回路を構築し、理解するというコンセプトの下で、iGEMはこれまで発展し続け、様々な遺伝子パーツが生み出され、様々な遺伝子回路が組めるようになった。事実、Parts Registryにそのコーディングシーケンスとなるパーツがある多様なタンパク質――色々な刺激に応答して転写を制御するものや、種々の物質を生合成する酵素、生産物を外部に分泌する輸送タンパク質など――と、そのタンパク質と特異的に相互作用する塩基配列を組み合わせて、大腸菌をはじめとするChassisに導入することで多様性に富んだ組み換え生物が作られてきた。
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===Oscillation===
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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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<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">
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<img src="https://static.igem.org/mediawiki/2013/a/a0/Kyoto_RNA_Prezi.png"/>
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</iframe>
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</center>
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</html>
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　しかし、遺伝子回路を設計するにあたって、タンパク質を用いて実現することが難しいような状況が現れることがある。タンパク質を設計するのは現在まだとても困難であり、特定の分子と特異的に相互作用させようとしたり、狙った部分の転写を調節しようとしたりすることにはまだ多くの技術的な壁がある。また、タンパク質は転写、翻訳、フォールディング、そして修飾という複数のステップを経て合成され、また分解にもある程度の時間がかかる。そのため、発現するタンパク質の種類を変えるときには、転写調節から発現されているタンパク質の量が完全に入れ替わるまでのタイムラグをある一定の時間(その長さはタンパク質の種類に依存するだろう)より短くすることは難しいと考えられる。
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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.
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[[File:Kyoto_RNA_Prezi.png]]
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</div>
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<div id="reportertab">
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　そこで今回我々は遺伝子回路の構成要素のタンパク質に代わるもう一つの候補として、転写制御因子や、蛍光によるレポーターとなるRNAを用いることを提案する。RNAを用いることのメリットは以下の二つである。
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===Repressor===
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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].
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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
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[[File:2013IGKUprojectRNArepressionMECHANISM.png]]
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[[File:2013IGKUprojectRNArepressionMECHANISM2.png]]
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</div>
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<div id="repressiontab">
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　RNA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。
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===Activator===
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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.
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[[File:No-binding-of-tetR-aptamer.png]][[File:Binding-of-tetR-aptamer.png]]
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</div>
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<div id="fusiontab">
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　さらにRNAは転写後、翻訳の時間を経ずにフォールディングが始まるため、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速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を適宜使い分けることで、全体としてのかかる時間幅を設計することもできるようになるかもしれない。
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===Reporter===
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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>
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We strongly suggest Spinach aptamer as a reporter of RNA.
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[[File:SPINACHの説明.png]]
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　今年、我々はRNAを転写制御因子やレポーターとして利用して遺伝子回路を構築できることを示すため、目標としてオシレーターの製作を掲げた。オシレーターは生物にとって重要な回路であり、また転写の抑制、促進の双方を満たすRNAが含まれるため、それらを以降の応用への例として挙げる。これからの合成生物学の発展のため、より多様性に富んだより複雑な遺伝子回路を設計するために、この技術がより広く用いやすくなることを望む。
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===Fusion===
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<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.
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We estimated the RNA structure to check whether or not unindicatd duplex is formed by open tool.
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</p>
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</div>
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<div id="conctab">
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== Oscillator Design ==
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==Experiment==
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<html><center><iframe 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"></iframe></center></html>
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After we constructed functional RNA generator, we checked the transcription of the RNA parts. To confirm this, we performed RT-PCR.<br>
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samples are following:<br>
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Negative control<br>
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*Non-promoter: Spinach-DT
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Experimental group<br>
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[[File:唯一のexperiment.png]]<br>
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We also checked whether fusion RNA we designed functions or not considering secondary structure with Centroid Fold[6]
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</div>
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== Activator ==
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==Result==
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=== Description ===
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===RT-PCR===
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　転写制御を行うような機能性RNAの例として、我々はtetR aptamerを挙げる。tetRはtetracyclineオペロンを構成するタンパク質であり、tetracycline非存在下でtetオペレータに結合し転写を抑制している。tetRはtetracyclineと結合することにより転写抑制を解除する。つまり、tetオペレータ下流の遺伝子は、tetracyclineの存在下でActivateされるということである。大腸菌内に存在する様々なsRNA (small noncoding RNA) の中には、<22-nucleotides-long fragmentsでtetracyclineと同等の働きを持つものが見つかっている。 (Hunsicker, 2009) 我々はこれを用いて、Activationの回路を構成した。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。
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We performed RT-PCR to confirm transcription of TetR aptamer(left) and Spinach(center).<br>
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[[File:ElectrophoresisRT1.png]]
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[[File:ElectrophoresisRT2.png]]
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===Structure Prediction===
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[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]
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[[File:antisense_spinach.png]]with Centroid Fold*
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<br>
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==Conclusion==
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We confirmed the transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP by using RT-PCR method.<br>
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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>
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We got ready for the construction of the oscilator circuit in wet lab.<br>
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<div id="futuretab">
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=== Assay ===
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==Future work==
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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>
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1. qualitative assay TatR aptamer<br>
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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>
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3, qualitatively Spinach assay (visual recognition & fluorescence microscopes)<br>
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We will check that DFHBI fluorescence on a plate with Spinach.<br>
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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>
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After that, we will substitute the values for oscilation model and try to solve simulate. Moreover we will continue assaying of our parts.<br>
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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>
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Finaly, we compare results of wet lab and dry lab and discuss a point in common/difference between the results.
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</div>
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<div id="achievetab">
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tetR aptamerがtetRの機能を抑制し、tetプロモータ下流の転写を促進するかを確認するために、次のような実験を行った。
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</div>
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実験群は、tetR aptamerを恒常的に発現する系である。対照標準として、以下を用意する。
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<div id="partslisttab">
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positive
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== Parts List ==
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*constitutive promoter-GFP。レポーターであるGFPがそのままで光るという機能確認。
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<groupparts>iGEM013 Kyoto</groupparts>
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*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。
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</div>
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<div id="referencetab">
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negative
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== Reference ==
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*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。
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[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>
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*tetR aptamer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。
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[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>
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[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>
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experimental group:<br>
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[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>
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1:Pcon-RBS-tetR-DT Ptet-RBS-GFP-DT Pcon-tetRaptamer-DT<br>
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[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>
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positive control:<br>
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[6][http://www.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]<br>
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2:Ptet-RBS-GFP-DT<br>
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3:Pcon-RBS-GFP-DT<br>
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negative control:<br>
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4:Pcon-RBS-tetR-DT Ptet-RBS-GFP-DT Pcon-anti_attenuator<br>
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5:Pcon-RBS-tetR-DT Ptet-RBS-GFP-DT Pcon-attenuator<br>
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6:Pcon-RBS-tetR-DT Ptet-RBS-GFP-DT Pcon-spinach<br>
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7:Pcon-RBS-tetR-DT Ptet-RBS-GFP-DT<br>
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=== Result ===
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Result fig
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== Replessor ==
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=== Description ===
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生体内でのゲノムの転写制御は様々な仕方によってなされる。その中には、タンパク質による制御だけでなく、RNAによるsystemも見つかっている。これは様々な生物で見つかっており、RNAワールド仮説に則れば古いシステムの名残かもしれないとも言われている(Corbino KA et al, 2005; Winkler et al., 2002)。
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例えば、Gram-negative bacteria Staphylococcus aureusのpT181と呼ばれるplasmidなどのコピー数のregulationには、RNAが関わっている。これは、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)。
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われわれはこれをRepressionの回路とした。
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=== Assay ===
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=== Result ===
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== Reporter ==
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=== Description ===
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　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、大きな速度でオシレーションするRNAの発現量を正確に反映することが出来る。
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　spinachの説明、Assayが中国のチームのパーツの機能追試であるということを述べる？
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=== Assay ===
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Experimental:
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*Pcon-spinach-DT
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*Pcon-antisense-spinach-DT
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Negative control
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*none
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*Pcon-tetR aptamer-DT
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*Pcon-tetR antisense
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*Pcon-tetR attenuator
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固定して検鏡(ヘキストとDFHBIの両方で染色)
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=== Result ===
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== Fusion ==
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=== Description ===
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実際にこれらを使って実験するとき、各Moduleを同時に使わなければいけないことは十分にありうる。転写抑制の様子をレポートする、因子Aで促進されBで抑制されるような系を作るなど。しかし、これをする上で問題となってくるのが、連結したとき相互作用や立体構造の問題によりそれぞれの機能が確認されないことである。
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タンパク質であれば、その問題を予測するのは難しい。しかし、RNAであれば、配列情報からかんたんに構造を予測し起こりうる問題を回避できる。
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われわれは、機能を確認したtetR, Antisense-Attenuator RNA, Spinach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。
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=== Assay ===
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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、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。
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1: Pcon-RBS-tetR-DT Ptet-RBS-GFP-DT Pcon-attenuator-tetRaptamer-DT<br>
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2: Pcon-attenuator-RBS-GFP-DT Pcon antisense<br>
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=== Structure Prediction ===
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Centroid fold, mfoldのfig
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[Result]
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各パーツは干渉することなく機能した。
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== Future view ==
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=== Description ===
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これらのRNAを用いた機構を組み合わせて、我々はひとつの回路を提案したい。
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<オシレーション回路の図>
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RNA ModuleによるActivation, repressionの機構を組み合わせ、Spinachで蛍光をみる、点滅する大腸菌が作れる。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予測される。
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=== Method ===
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この回路が実際に働くことを示すため、我々はコンピューターシュミレーションを行った。
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<こうこうこういう式で…こうこうこういうプログラム組んで…>
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=== Result ===
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<Modelingの結果の図とか出せるの…かな?>
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== References ==
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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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