Introduction

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. 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.

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.

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.

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 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.

Activator

We took up TetR aptamer as an example of functional RNA which induces a transcription. TetR aptamer induces tetracycline promoter (Ptet) by binding to tetracycline repressor (TetR), which represses Ptet. When TetR aptamer binds to TetR, induces conformational change of TetR. As a result, TetR cannot come to bind to tetracycline operator (tetO). We ordered MBL=IDT gene synthesis of pT181 attenuator region DNA, antisense DNA and TetR aptamer with prefix and suffix. We transferred these parts to pSB1C3 and constructed device for antisense and attenuator assay (Fig. ).

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。

Spinach is an example of a reporter RNA aptamer，which emits the green fluorescence like GFP when it binds to a fluorophore (DFHBI), which is a derivative fluorophore of GFP. DFHBI doesn't emit fluorescence alone. That is to say, if fluorescence is observed after DFHBI is added into liquid culture, it is manifest that Spinach is expressed. If Spinach exists, it combines with DFHBI and DFHBI emits fluorescence. Hence, by using Spinach, it’s possible not only to image RNA directly, but also accurately reflect the transcription level, which can’t be confirmed via stable protein because RNA is degraded faster than protein.

We strongly suggest Spinach aptamer as a reporter of RNA.

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 formed by open tool using computer.

Experiment

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

• Spinach-DT

Experimental group

We also check whether fusion RNA designed by us functions or not considering secondary structure with Centroid Fold[5]

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. qualitatively assay tatR aptamer
To confirm the act of tetR aptamer inducing Ptet ,we allow IPTG-inducble tetR aptamer to derive GFP on plate. As negative controls, we use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we also use GFP.
After that, we will substitute the values for oscilation model and try to solve simulate. And 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 compair 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.ncrna.org/ Functional RNA Project provided by Computational Biology Research Center (CBRC)]