Revision as of 02:43, 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, 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, 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)

Fig. 1 Ptet - rbs - traR - tt (pSB6A1) Ptra - rbs - gfp - tt (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 were 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 and the number of this region is not enough for non-A. tumefaciens creatures 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

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      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Introduction</b></h1><!-- 小見出し -->
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">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 <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">TraI Assay</a> page). Therefore, we searched the way to detect that C8 was synthesized by <span style="font-style: italic">E. coli</span> introduced C8 synthesis gene, traI. This section describes the evaluation of Tra system (a kind of Quorum Sensing derived from <span style="font-style: italic">Agrobacterium tumefaciens</span>).
+     <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">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 <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">"TraI Assay"</a> page). Therefore, we searched the way to detect that C8 was synthesized by <span style="font-style: italic">E. coli</span> introduced C8 synthesis gene, traI. This section describes the evaluation of Tra system (a kind of Quorum Sensing derived from <span style="font-style: italic">Agrobacterium tumefaciens</span>).
 </p>