Revision as of 03:02, 1 November 2017

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iGEM Tokyo Tech

TraR Reporter Assay

Introduction

In our project, we use a kind of Quorum Sensing signaling molecules (Acyl-homoserine lactones, AHLs), 3OC8HSL (hereafter C8) as a signaling molecule of cross-kingdom communication from bacteria to human cells (Read "TraI Assay" page). Therefore, we searched the way to detect that C8 was synthesized by E. coli introduced C8 synthesis gene, from traI. This section describes the evaluation of Tra system (a kind of Quorum Sensing derived from Agrobacterium tumefaciens).

Summary

The purpose of the experiment is to evaluate the reporter gene of Tra system, from traR works correctly in E. coli cells. We constructed the two plasmids shown below and introduced them to E. coli. To the end, adding C8 into the E. coli and investigate TraR-C8 complex activate transcription from Ptra.

- Plasmids

Sample(Fig. 1)

A. Ptet – rbs – traR (pSB6A1)

B. Ptra – rbs – gfp (pSB3K3)

Negative control (Fig. 2)

C. pSB6A1

D. pSB3K3

Results

The results showed that the value of RFU of GFP / Turbidity of samples and that of negative controls was same. This indicated that GFP expression was not induced by TraR-C8 complex.

Fig. 3 RFU of GFP / Turbidity of TraR Reporter Assay

Discussion

From the results, Tra reporter system did not work in E. coli. This is because the tra box where TraR-C8 complex binds upstream Ptra is essential to active transcription(Read Chimeric Transcription Factor Assay page) and the number of this region is not enough for non-A. tumefaciens organisms to activate transcription. From other our experiment, C8 could be detect by Lux system (a kind of Quorum Sensing system derived from Vibrio fischeri). Thus, we decided to use Lux system to detect that C8 was synthesized by E. coli introduced traI (Read TraI Assay page)

Reference

[1] Foundational Platform for Mammalian Synthetic Biology, 2012 Noah Davidsohn et.al

[2]Conserved cis-Acting Promoter Elements Are Required for Density-Dependent Transcription of Agrobacterium tumefaciens Conjugal Transfer Genes, 1995 CLAY FUQUA et.al

@@ Line 148: / Line 148: @@
 <center><figure>
      	<img src="https://static.igem.org/mediawiki/2017/1/14/T--TokyoTech--TraRAssay.png" style="max-width:60%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 1  Ptet - <span style="font-style: italic">rbs - traR - tt</span> (pSB6A1) Ptra - <span style="font-style: italic">rbs - gfp - tt</span> (pSB3K3)</figcaption>
+     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 1 Construction of sample</figcaption>
          </figure></center></br>
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 <center><figure>
      	<img src="https://static.igem.org/mediawiki/2017/8/8b/T--TokyoTech--Plasmid_Nega.png" style="max-width:60%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 2  pSB6A1 pSB3K3</figcaption>
+     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 2  Construction of Negative control</figcaption>
          </figure></center>
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      <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"><p style="text-indent:1em">From the results, Tra reporter system did not work in <span style="font-style: italic">E. coli</span>. This is because the tra box where TraR-C8 complex binds upstream Ptra is essential to active transcription(Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/Chimeric_Transcription_Factor">Chimeric Transcription FactorAssay</a> page) and the number of this region is not enough for non-<span style="font-style: italic"><span style="font-style: italic">A. tumefaciens</span></span> organisms to activate transcription. From other our experiment, C8 could be detect by Lux system (a kind of Quorum Sensing system derived from <span style="font-style: italic">Vibrio fischeri</span>). Thus, we decided to use Lux system to detect that C8 was synthesized by <span style="font-style: italic">E. coli</span> introduced traI (Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">TraI Assay</a> page)
+     <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">From the results, Tra reporter system did not work in <span style="font-style: italic">E. coli</span>. This is because the tra box where TraR-C8 complex binds upstream Ptra is essential to active transcription(Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/Chimeric_Transcription_Factor">Chimeric Transcription Factor Assay</a> page) and the number of this region is not enough for non-<span style="font-style: italic"><span style="font-style: italic">A. tumefaciens</span></span> organisms to activate transcription. From other our experiment, C8 could be detect by Lux system (a kind of Quorum Sensing system derived from <span style="font-style: italic">Vibrio fischeri</span>). Thus, we decided to use Lux system to detect that C8 was synthesized by <span style="font-style: italic">E. coli</span> introduced traI (Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">TraI Assay</a> page)