Team:Kyoto/projectRNA

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=RNA Module=
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<ul class="Kyoto-toptab">
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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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<div id="projectRNA">
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=RNA Oscillator=
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<div id="introtab">
==Introduction==
==Introduction==
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<p>1遺伝子編集の技術が確立されて以降、遺伝子転写制御回路の様々なアイデアが出され、その多くは実現されてきた。iGEMも例外でなく、現在もとても多様な遺伝子回路が世界中の学生達の手によって作られ続けている。そのほとんどは、機能要素としてタンパク質のみを用いてきた。実際、長年研究者たちが追い続けていた生物の持つ機能性分子はそのほとんどがタンパク質であった。そのため、遺伝子回路の要素として使用可能な状態になったタンパク質は多く、手法への経験や知見も溜まっている。</p>
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===Motivation===
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<p>After methods of gene engineering was established, many researchers hit on various ideas of synthetic regulatory systems for gene expression and most of these ideas are actually constructed. Even now, various and novel systems are still being made by researchers and iGEMers and most of them are designed with proteins as functional modules. Proteins are studied for a long time. Therefore a lot of varieties of proteins are available, and experience and knowledge of the methods with proteins in gene circuit are well progressed.</p>
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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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<p>2しかし、遺伝子回路を設計するにあたって、希に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。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。</p>
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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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<p>However, it may sometimes be difficult to use protein for constructing transcriptional regulation system. There may be technical difficulties for designed proteins to interact with the specific molecule and to regulate transcription of the target locus except the appropriate promoter. Proteins function only after processing transcription, translation, folding and modification. In addition, degradation of protein takes a fixed time.
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</div>
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Therefore, when we regulate the expression of the protein, it takes time for the protein already expressed to degrade, and a newly expressed protein to process transcription, translation, and folding. This time lag seems to be difficult to be shortened than a fixed time which depends on the kind of protein.</p>
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<div id="activationtab">
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<p>3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、転写制御因子となるRNAを用いることを提案する。機能性RNAはまだ研究され始めたばかりではあるが、今後その研究はますます発展することが予想される。今後転写制御系の構成要素となり得る機能性RNAは増えていくだろう。RNAを用いることのメリットは以下の二つである。</p>
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<p>Therefore, in order to overcome this weak point, we propose designing synthetic regulatory systems for gene expression with functional RNA instead of protein. Though functional RNA has just begun to be studied, we can expect a remarkable development. And RNA applied for transcriptional regulator should keep on increasing. The merits to use RNA modules are the following two reasons.</p>
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<p>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。</p>
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<p>Compared to protein, RNA is easier to predict its secondary structure, and to design it 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.</p>
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<p>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。 </p>
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<p>Functional RNA works while it folds and can make its folding without the need of translation. Also RNA degradation in vivo is faster than protein. So it is suggested that transcriptional regulation can response faster using RNA Module. Therefore when we use both protein and RNA, we can control how long transcriptional regulation response.</p>
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<p>6転写制御系を設計する上で必要なパーツは、遺伝子の転写をはじめるアクチベーター、遺伝子の転写をとめるリプレッサーである。その2つの役割を果すRNAとして、我々はTetRアプタマー、Attenuator regionとAntisense RNAを選び、それらが転写制御の役割を果たすことを確認した。また、構造予測が比較的簡単に出来る特徴を利用し、これらの機能性RNAを活性部位の立体構造に影響しないように互いに繋げあわせたものを設計し、活性に影響が出ないことを実験により確認した。</p>
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<p>The essential parts for synthetic regulatory systems are activator and repressor. The activator stimulates the transcription of the gene and the repressor blocks the transcription. We selected TetR aptamer and Attenuator region with Antisense RNA as activator and repressor. Firstly, we confirmed that these 2 RNA modules functions really work. Moreover, utilizing the property of RNA that it can be predicted secondary structure easily, we designed fusion RNA modules being careful to do not effect the secondary structure of each other. Then we confirmed that these fusion RNA modules.</p>
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【メモ:figが必要】
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==Activation==
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===Oscillation===
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<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遺伝子の発現を見、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。)
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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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<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 group in expression of GFP.</p>
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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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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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===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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===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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===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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===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.
</p>
</p>
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<p>-----コンストラクション------</p>
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</div>
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Positive Control<br>
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<div id="conctab">
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A. Ptet-GFP<br>
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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。<br>
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Negative Control<br>
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B. Ptet-GFP, Pcon-TetR<br>
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-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>
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C-E. Ptet-GFP, Pcon-TetR, Pcon-RNAs(anti_attenuator, attenuator, spinach)<br>
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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。<br>
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Experimental Group<br>
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F. Ptet-GFP, Pcon-TetR, Pcon-tetRaptamer<br>
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<br>
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Positive Control<br>
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<br>
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Ptet-GFP<br>
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<p>In order to check whether the GFP gene is expressed when there is no tet Protein, we introduced only Ptet-GFP.</p>
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Negative Control<br>
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<br>
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Ptet-GFP, Pcon-tetR<br>
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<p>Through this E.coli, we can confirm that when tetR is expressed, tetR surpress the expression of GFP.</p>
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C-E. Ptet-GFP, Pcon-tetR, Pcon-RNAs(anti_attenuator, attenuator, spinach)<br>
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<p>These kinds of E.coli shows that the surpression of the function of tetR is caused only by tetRaptamer.</p>
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Experimental Group<br>
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<br>
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Ptet-GFP, Pcon-tetR, Pcon-tetRaptamer<br>
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<p>-----コンストラクション------</p>
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<p>-----fig Caption------</p>
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<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>
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<p>-----fig Caption------</p>
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<p>顕微鏡による観察とqRT-PCRによりtetRタンパク質を発現させた場合、GFPの発現量が減っていると分かるので、tetRタンパク質がtetプロモーター下流のGFPの発現を抑制していることが分かる。顕微鏡による観察とqRT-PCRによりtetRタンパク質がある時、tetRアプタマーが発現された場合のみGFPの発現量が増えていると分かるので、GFP上流にあるtetプロモーターを抑制していたtetRタンパクをtetRアプタマーが抑制した、つまりtetRアプタマーはtetRタンパク質存在下でtetプロモーター下流に対しアクチベーターとして働くと言える。</p>
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==Repression==
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==Experiment==
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<p>転写の抑制を行うようなRNAの例として、我々は、ゲノムDNA鎖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)。
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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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われわれはこれをRepressionの回路とした。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>
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samples are following:<br>
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<p>------const------</p>
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Negative control<br>
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Positive Control<br>
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*Non-promoter: Spinach-DT
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A-C: Pcon-RNAs(tetRaptamer, attenuator, antisense), Pcon-atte-GFP<br>
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Experimental group<br>
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-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>
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[[File:唯一のexperiment.png]]<br>
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D: Pcon-atte-GFP<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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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>
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</div>
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Experimental Group<br>
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E: Pcon-antisense Pcon-atte-GFP<br>
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<p>------const------</p>
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<p>---figcaption----</p>
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<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>
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<p>---figcaption----</p>
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<p>顕微鏡による観察とqRT-PCRによりAttenuator antisense RNAを発現させた場合のみ、Attenuator Region下流にあるGFPの発現量が減ったことが分かった。このことからAttenuator antisense RNAはAttenuator Region下流に対して抑制効果があり、リプレッサーとして働くと言える(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>
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==<s>Reporter</s>==
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==Result==
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<s>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>
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===RT-PCR===
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【メモ:Assay、Result、Discussion】</s>
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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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==Fusion==
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===Structure Prediction===
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<p>実際にこれらを使って遺伝子回路を構築することを考えると、複数のModuleを同じ要素の中に含めなければならない状況が発生することは十分に有り得る。転写抑制の様子をレポートするとき、因子Aで促進されBで抑制されるような系を作るときなどである。この融合を行う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阻害されるのではないかということである。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、配列情報から比較的簡単に二次構造を予測することができ、起こりうる問題を予見し回避できる。われわれは、機能を確認したtetR aptamer, Antisense-Attenuator RNA, <s>Spinach</s>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(a)、<s>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。</s></p>
+
[[File:2013IGKUprojectRNAfusionCENTROIDattenuatoraptamer.png]]
-
<p>-----const-------</p>
+
[[File:antisense_spinach.png]]with Centroid Fold*
-
experimental group<br>
+
<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>
+
-
<p>------const-------</p>
+
-
<p>We used centroid fold (URL) and mfold (URL) to predict the secondary structure of RNA(a). As the picture shown in below, the structure of tetR aptamer is not effected by attenuator stem loop. This suggests that the efficiency of tetR aptamer is not effected by the existence of attenuator stem loop.</p>
+
-
<p>Centroid fold, mfoldのfig-tetR aptamer only----tetR aptamer-antisence</p>
+
-
<p>蛍光顕微鏡の観察とQRTによりtetRアプタマーRNAが発現していないときより、GFPは多く発現していると分かるのでtetRアプタマーgene以外にattenuator Region gene がある場合でもattenuator Region RNAに妨害されることなくtetRアプタマーRNAはアクチベーターとして働くと言える
+
-
蛍光顕微鏡の観察とQRTによりattenuator RNA gene以外にspinach RNA geneがあってもAttenuator Region下流のGFP geneの発現量の減りが確認されたので、attenuator RNAはspinach RNAがあっても妨害されることなく、Attenuator Region下流に対してリプレッサーとして働くと言える。
+
-
蛍光顕微鏡による観察でDFHBI存在下で蛍光が確認されることからspinach RNAはattenuator RNAがあっても妨害されずに、レポーターとして働くと言える。</p>
+
-
【memo result書き直し】
+
==Conclusion==
==Conclusion==
-
我々は以上の実験で、Activator, Repressor、Reporterの機能を持つ各機構の確認とそれぞれを繋ぎあわせたものの構造予測、および機能確認をした。
+
We confirmed the transcription of TetR aptamer, antisense-Spinach, Spinach, and GFP by using RT-PCR method.<br>
-
==Future Work==
+
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>
-
より複雑な遺伝子回路の一例として、これらのModuleを用いて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が示唆される。ここで、その例として、Spinach蛍光の発現量を振動させるオシレーターを上げたい。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、以降の応用への例として適切であると考えられる。<br>
+
We got ready for the construction of the oscilator circuit in wet lab.<br>
-
私達は、次のようなオシレーションの回路を提案する。<br>
+
<div id="futuretab">
-
<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><br>
+
 
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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。<br>
+
==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>
 +
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>
 +
Finaly, we compare results of wet lab and dry lab and discuss a point in common/difference between the results.
 +
</div>
 +
<div id="achievetab">
-
==Achievement==
+
</div>
-
我々は、このプロジェクトで以下のことを達成した。<br>
+
<div id="partslisttab">
-
①<br>
+
-
②<br>
+
-
③<br>
+
-
④<br>
+
-
⑤<br>
+
-
⑥<br>
+
== Parts List ==
== Parts List ==
-
-自分たちでつくったもの<br>
+
<groupparts>iGEM013 Kyoto</groupparts>
-
iGEMの仕様のやつのせてね<br>
+
</div>
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-他チームのを機能確認したもの<br>
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<div id="referencetab">
-
tetR<br>
+
-
spinach<-光ってねーんだよ起きろ<br>
+
== Reference ==
== Reference ==
-
a
+
[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>
 +
[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>
 +
[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>
 +
</div>
 +
</div>
{{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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