Difference between revisions of "Team:TokyoTech/Experiment/Chimeric Transcription Factor"

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　In this assay, we investigated whether human cells (the EA.hy926 cell line) receive AHL, a signaling molecule that is synthesized in and exported from <span style="font-style: italic">E. coli</span> and induce the transcription of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes to synthesize iP(Read <a href = https://2017.igem.org/Team:TokyoTech/Experiment/AHK4_Assay>"AHK4 Assay"</a> page).
 
　In this assay, we investigated whether human cells (the EA.hy926 cell line) receive AHL, a signaling molecule that is synthesized in and exported from <span style="font-style: italic">E. coli</span> and induce the transcription of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes to synthesize iP(Read <a href = https://2017.igem.org/Team:TokyoTech/Experiment/AHK4_Assay>"AHK4 Assay"</a> page).
 
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　As shown in Fig. 1, two plasmids were introduced into the EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses RelA/NLS/TraR. When RelA/NLS/TraR binds to C8, this complex binds to the (tra box)<sub>7</sub> sequence and activates transcription from the CMV minimal promoter (CMV min). The (tra box)<sub>7</sub> sequence is the binding site for TraR (and also RelA/NLS/TraR) and works as the transcription enhancer. The core sequence of the (tra box)<sub>7</sub> is “ATGTGCAGATCTGCACAT” and this sequence is repeated seven times. As a result, the <span style="font-style: italic">atIPT4</span>
 
　As shown in Fig. 1, two plasmids were introduced into the EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses RelA/NLS/TraR. When RelA/NLS/TraR binds to C8, this complex binds to the (tra box)<sub>7</sub> sequence and activates transcription from the CMV minimal promoter (CMV min). The (tra box)<sub>7</sub> sequence is the binding site for TraR (and also RelA/NLS/TraR) and works as the transcription enhancer. The core sequence of the (tra box)<sub>7</sub> is “ATGTGCAGATCTGCACAT” and this sequence is repeated seven times. As a result, the <span style="font-style: italic">atIPT4</span>
 
 and <span style="font-style: italic">log1</span> genes are transcribed depending on C8. The <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes are derived from <span style="font-style: italic">A. thaliana</span>, and their products jointly work as iP synthetase.
 
 and <span style="font-style: italic">log1</span> genes are transcribed depending on C8. The <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes are derived from <span style="font-style: italic">A. thaliana</span>, and their products jointly work as iP synthetase.
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       <figure>
 
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       <img src="https://static.igem.org/mediawiki/2017/1/15/T--TokyoTech--human_cell_circuit.png" style="max-width:75%">
 
       <img src="https://static.igem.org/mediawiki/2017/1/15/T--TokyoTech--human_cell_circuit.png" style="max-width:75%">
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       <figcaption style="font-family: Poppins;font-size: 16px">Fig1. Construction of iP synthetase genes</figcaption>
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       <figcaption style="font-size: 16px">Fig. 1 Construction of iP synthetase genes</figcaption>
 
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　IVS (intervining sequence) is one kind of introns and is important for increasing the mRNA stability in eukaryotic cells. IRES (internal ribosome entry site) is an RNA element that allows translation initiation in a cap-independent manner. The term “polyA” indicates the polyadenylation signal that  is important for the nuclear export, translation, and stability of mRNA.
 
　IVS (intervining sequence) is one kind of introns and is important for increasing the mRNA stability in eukaryotic cells. IRES (internal ribosome entry site) is an RNA element that allows translation initiation in a cap-independent manner. The term “polyA” indicates the polyadenylation signal that  is important for the nuclear export, translation, and stability of mRNA.
 
  </p>
 
  </p>
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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 -->
 
     <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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　The transformed cells were treated or not treated with different concentrations of C8. Then, the mRNA level of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> was analyzed using quantitative RT-PCR. As shown in Fig. 2, the CMV minimal promoter was activated following the addition of C8.  
 
　The transformed cells were treated or not treated with different concentrations of C8. Then, the mRNA level of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> was analyzed using quantitative RT-PCR. As shown in Fig. 2, the CMV minimal promoter was activated following the addition of C8.  
  
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     <img src="https://static.igem.org/mediawiki/2017/e/e9/Human_cell_result_v3.png" style="max-width:85%">
 
     <img src="https://static.igem.org/mediawiki/2017/e/e9/Human_cell_result_v3.png" style="max-width:85%">
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     <figcaption style="font-family: Poppins;font-size: 16px">Fig. 2 Result of the qualitative experiment</figcaption>
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     <figcaption style="font-size: 16px">Fig. 2 Result of the qualitative experiment</figcaption>
 
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　The term“Cont”means the control cells that are not electroporated, while “EP” means the electroporated cells. The concentrations of added C8 are indicated below the bars.  
 
　The term“Cont”means the control cells that are not electroporated, while “EP” means the electroporated cells. The concentrations of added C8 are indicated below the bars.  
 
  </p>
 
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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h1>
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h1>
 
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　We confirmed that the transcription of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes were induced by C8 addition and the degree of induction depends on C8 concentration.
 
　We confirmed that the transcription of <span style="font-style: italic">atIPT4</span> and <span style="font-style: italic">log1</span> genes were 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>
 
     <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"><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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     <figcaption style="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  
 
4: 159-165.</p>
 
4: 159-165.</p>
 
<div style="margin-top:16px">
 
<div style="margin-top:16px">

Revision as of 00:20, 2 November 2017

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iGEM Tokyo Tech

Chimeric Transcription Factor Assay

Introduction

　In this assay, we investigated whether human cells (the EA.hy926 cell line) receive AHL, a signaling molecule that is synthesized in and exported from E. coli and induce the transcription of atIPT4 and log1 genes to synthesize iP(Read "AHK4 Assay" page).
　AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing”(Read "TraI Assay" page). Among several kinds of AHLs, 3OC8HSL (C8) was chosen in the assay.

Summary

　As shown in Fig. 1, two plasmids were introduced into the EA.hy926 cells by electroporation. The CAG promoter (pCAG) constantly expresses RelA/NLS/TraR. When RelA/NLS/TraR binds to C8, this complex binds to the (tra box)7 sequence and activates transcription from the CMV minimal promoter (CMV min). The (tra box)7 sequence is the binding site for TraR (and also RelA/NLS/TraR) and works as the transcription enhancer. The core sequence of the (tra box)7 is “ATGTGCAGATCTGCACAT” and this sequence is repeated seven times. As a result, the atIPT4  and log1 genes are transcribed depending on C8. The atIPT4 and log1 genes are derived from A. thaliana, and their products jointly work as iP synthetase.

Fig. 1 Construction of iP synthetase genes
　IVS (intervining sequence) is one kind of introns and is important for increasing the mRNA stability in eukaryotic cells. IRES (internal ribosome entry site) is an RNA element that allows translation initiation in a cap-independent manner. The term “polyA” indicates the polyadenylation signal that is important for the nuclear export, translation, and stability of mRNA.

Results

　The transformed cells were treated or not treated with different concentrations of C8. Then, the mRNA level of atIPT4 and log1 was analyzed using quantitative RT-PCR. As shown in Fig. 2, the CMV minimal promoter was activated following the addition of C8.

Fig. 2 Result of the qualitative experiment
　The term“Cont”means the control cells that are not electroporated, while “EP” means the electroporated cells. The concentrations of added C8 are indicated below the bars.

Discussion

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

Protocol

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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