Difference between revisions of "Team:TokyoTech/Experiment/Human Cell Assay"

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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Overview</b></h1><!-- 小見出し -->
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     <p style="font-family: Poppins;font-size: 16px">概要をここに
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     <p style="font-family: Poppins;font-size: 16px">In this assay, we investigated whether human cells (<span style="font-style: italic">EA.hy926</span> cell) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atlPT4 and log1 genes to synthesize iP.
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    <p style="font-family: Poppins;font-size: 16px"><u>Note</u></p>
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    <p style="font-family: Poppins;font-size: 16px">AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”. Among several kinds of the signaling molecules, we used 3OC8HSL(C8) in the assay.</p>
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<div class="w3-container" id="summary" style="margin-top:20px"><!-- この箱の中にテキストや画像をまとめる -->
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    <p style="font-family: Poppins;font-size: 16px">As shown in Fig.1, two kinds of constructs were introduced into <span style="font-style: italic">EA.hy926</span> cells by electroporation. The CAG promoter (pCAG) constantly expresses the chimeric protein, relA/NLS/traR. When relA/NLS/traR binds with C8, this complex binds to (tra box)7 sequence (the enhancer sequence; see below) and activates transcription from the CMV minimal promoter (CMV min). As a result, at the lPT4 and log1 genes are transcribed depending on C8. The <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> gene products jointly work as IP synthetase.
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      <img src="https://static.igem.org/mediawiki/2017/1/15/T--TokyoTech--human_cell_circuit.png" style="max-width:50%">
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      <figcaption style="font-family: Poppins;font-size: 16px">Fig1. Construction of IP synthetase gene</figcaption>
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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Results</b></h1><!-- 小見出し -->
 
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     <figcaption style="font-family: Poppins;font-size: 16px">Fig2. Result of the qualitative experiment</figcaption>
 
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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h1>
 
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     <p style="font-family: Poppins;font-size: 16px">考察
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     <p style="font-family: Poppins;font-size: 16px">We confirmed that the transcription of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes are induced by C8 addition and the degree of induction depends on C8 concentration.
 
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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Reference</b></h1>
 
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     <p style="font-family: Poppins;font-size: 16px">参考文献
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     <p style="font-family: Poppins;font-size: 16px"><p>Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL
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4: 159-165.</p>
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Latest revision as of 09:25, 30 October 2017

<!DOCTYPE html> Coli Sapiens

iGEM Tokyo Tech

Human Cell Assay

Introduction

In this assay, we investigated whether human cells (EA.hy926 cell) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atlPT4 and log1 genes to synthesize iP.


Note

AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”. Among several kinds of the signaling molecules, we used 3OC8HSL(C8) in the assay.

Summary

As shown in Fig.1, two kinds of constructs were introduced into EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses the chimeric protein, relA/NLS/traR. When relA/NLS/traR binds with C8, this complex binds to (tra box)7 sequence (the enhancer sequence; see below) and activates transcription from the CMV minimal promoter (CMV min). As a result, at the lPT4 and log1 genes are transcribed depending on C8. The atIPT4 and log1 gene products jointly work as IP synthetase.

Fig1. Construction of IP synthetase gene

Results

文章

Fig2. Result of the qualitative experiment

Discussion

We confirmed that the transcription of atIPT4 and log1 genes are induced by C8 addition and the degree of induction depends on C8 concentration.

Reference

Petra Neddermann, Cesare Gargioli, Ester Muraglia, Sania Sambuncini, Fabio Bonelli, Raffaele De Francesco, Riccardo cortese (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription facter TraR. EMBO reports VOL 4: 159-165.

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