Latest revision as of 09:25, 30 October 2017

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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.

@@ Line 62: / Line 62: @@
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px">In this assay, we checked whether human cells (<span style="font-style: italic">EA.hy926</span> cell) receive AHL, a signaling molecule derived from <i>E. coli</i> and induce the transcription of <i>atlPT4</i> gene and <i>log1</i> gene which synthesize iP. Also, we checked whether IP (Isopentenyl adenine) molecules are synthesized in <span style="font-style: italic">EA.hy926</span> cells by TLC.
+     <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.
-     </p>
+     </p>
      <br>
      <p style="font-family: Poppins;font-size: 16px"><u>Note</u></p>
-     <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 3OC8AHL(C8) in the assay.</p>
+     <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>
    </div>
@@ Line 76: / Line 77: @@
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px">As shown in Fig. , we introduced two kinds of constructs into <span style="font-style: italic">EA.hy926</span> cells by electroporation. CAG promoter constantly expresses relA/NLS/traR. After the protein binds with 3OC8AHL(C8), the complex exposes nuclear localization signal and activates CMVmin promoter in CMVmin_atIPT4_IVS_IRES_LOG1_polyA. As a result of pCMVmin's activation, downstream <i>atlPT4</i> gene and <i>log1</i> gene are transcripted. <i>atlPT4</i>  gene and <i>log1</i> gene jointly work as IP synthetase.
+     <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.
      </p>
@@ Line 82: / Line 84: @@
        <figure>
        <img src="https://static.igem.org/mediawiki/2017/1/15/T--TokyoTech--human_cell_circuit.png" style="max-width:50%">
-       <figcaption style="font-family: Poppins;font-size: 16px">Fig. 画像タイトル</figcaption>
+       <figcaption style="font-family: Poppins;font-size: 16px">Fig1. Construction of IP synthetase gene</figcaption>
        </figure>
        </div>
@@ Line 99: / Line 101: @@
      	<figure>
      	<img src="https://static.igem.org/mediawiki/2017/f/f9/T--TokyoTech--human_cell_results_1022.png" style="max-width:50%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 画像タイトル</figcaption>
+     	<figcaption style="font-family: Poppins;font-size: 16px">Fig2. Result of the qualitative experiment</figcaption>
      	</figure>
      	</div>
@@ Line 109: / Line 111: @@
      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h1>
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px">We checked the transcription of <i>atlPT4</i> gene and <i>log1</i> gene are induced by C8 addition and the induction depends on C8 concentration.
+     <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.
      </p>