Difference between revisions of "Team:TokyoTech/Experiment/TraR Reporter Assay"

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TraR Reporter Assay
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<h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center">TraR Reporter Assay</h1>
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<div class="w3-container" id="overview" 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"><p style="text-indent:1em">In our project, we use a kind of Quorum Sensing signaling molecules ([N-]acyl-homoserine lactones, AHLs), 3-oxo-C8 HSL (hereafter C8) as a signaling molecule of inter-kingdom communication from bacteria to human cells. 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>).
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    <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">The purpose of the experiment is to evaluate the reporter gene of Tra system, traR works correctly in <span style="font-style: italic">E. coli</span> cells. We constructed the two plasmids shown below and introduced them to <span style="font-style: italic">E. coli</span>. To the end, adding C8 into the <span style="font-style: italic">E. coli</span> and investigate TraR-C8 complex activate transcription from Ptra.
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    <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">- Plasmids</p>
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      <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">Sample</p>
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          <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">A. Ptet – rbs – traR (pSB6A1)</p>
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          <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">B. Ptra – rbs – gfp (pSB3K3)</p><br></br>
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      <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">Negative control</p>
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          <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">C. pSB6A1 </p>
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          <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">D. pSB3K3 </p>
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<p style="text-indent:1em"> </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 -->
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    <hr style="width:50px;border:5px solid red" class="w3-round">
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    <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">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.</p>
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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"><p style="text-indent:1em">From the results, Tra reporter system did not work in <span style="font-style: italic">E. coli</span> even though the temperature conditions were changed. 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-<span style="font-style: italic"><span style="font-style: italic">A. tumefaciens</span></span> 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 <span style="font-style: italic">E. coli</span> introduced traI (Read TraI Assay page)
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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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    <hr style="width:50px;border:5px solid red" class="w3-round">
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    <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">[1] Foundational Platform for Mammalian Synthetic Biology, 2012 Noah Davidsohn et.al
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<div class="w3-light-grey w3-container w3-padding-32" style="margin-top:75px;padding-right:58px"><p class="w3-right">Hajime Fujita:  <a href="96haji.me" title="W3.CSS" target="_blank" class="w3-hover-opacity">All Rights Reserved</a></p></div>
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Revision as of 13:35, 29 October 2017

<!DOCTYPE html> Coli Sapiens

iGEM Tokyo Tech

TraR Reporter Assay

Introduction

In our project, we use a kind of Quorum Sensing signaling molecules ([N-]acyl-homoserine lactones, AHLs), 3-oxo-C8 HSL (hereafter C8) as a signaling molecule of inter-kingdom communication from bacteria to human cells. 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

A. Ptet – rbs – traR (pSB6A1)

B. Ptra – rbs – gfp (pSB3K3)



Negative control

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.

Discussion

From the results, Tra reporter system did not work in E. coli even though the temperature conditions were changed. 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

Hajime Fujita: All Rights Reserved