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

 
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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 -->
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Introduction</b></h1><!-- 小見出し -->
 
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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), 3OC8HSL (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-size: 16px; 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, from <span style="font-style: italic">traI</span>. 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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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Summary</b></h1><!-- 小見出し -->
 
     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Summary</b></h1><!-- 小見出し -->
 
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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-size: 16px; text-indent:1em">The purpose of the experiment is to evaluate the reporter gene of Tra system, from <span style="font-style: italic">traR</span> 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-size: 16px; text-indent:1em">- Plasmids</p>
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       <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">Sample(Fig. 1)</p>
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       <p style="font-size: 16px">Sample(Fig. 1)</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-size: 16px">A. Ptet – <span style="font-style: italic">rbs – traR</span> (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-size: 16px">B. Ptra – <span style="font-style: italic">rbs – gfp</span> (pSB3K3) </p><br></br>
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     <img src="https://static.igem.org/mediawiki/2017/1/14/T--TokyoTech--TraRAssay.png" style="max-width:60%">
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     <figcaption style="font-size: 16px">Fig. 1 Construction of sample</figcaption>
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     <figcaption style="font-family: Poppins;font-size: 16px">Fig. 1 Ptet - rbs - traR - tt (pSB6A1) Ptra - rbs - gfp - tt (pSB3K3)</figcaption>
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           <p style="font-size: 16px">Negative control (Fig. 2)</p>
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1em">Negative control (Fig. 2)</p>
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           <p style="font-size: 16px">C. pSB6A1 </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-size: 16px">D. pSB3K3 </p>
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     <img src="https://static.igem.org/mediawiki/2017/8/8b/T--TokyoTech--Plasmid_Nega.png" style="max-width:60%">
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     <figcaption style="font-size: 16px">Fig. 2  Construction of Negative control</figcaption>
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     <figcaption style="font-family: Poppins;font-size: 16px">Fig. 2  pSB6A1 pSB3K3</figcaption>
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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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  <p style="font-size: 16px; text-indent:1em">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.</p>
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<figure>
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<center><figure>
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     <img src="https://static.igem.org/mediawiki/2017/a/a1/T--TokyoTech--TraR_reporter_assay.png" style="max-width:60%">
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     <img src="https://static.igem.org/mediawiki/2017/a/a1/T--TokyoTech--TraR_reporter_assay.png" style="max-width:80%">
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     <figcaption style="font-family: Poppins;font-size: 16px">Fig. 3 RFU of GFP / Turbidity of TraR Reporter Assay
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     <figcaption style="font-size: 16px">Fig. 3 RFU of GFP / Turbidity of TraR Reporter Assay
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         </figure></center>
  
  
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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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    <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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  <p style="font-size: 16px; 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)
  
  
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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 style="text-indent:1em">[1] Foundational Platform for Mammalian Synthetic Biology, 2012 Noah Davidsohn et.al
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    <p style="font-size: 16px; text-indent:1em">[1] Foundational Platform for Mammalian Synthetic Biology, 2012 Noah Davidsohn et.al</p>
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    <p style="font-size: 16px; text-indent:1em">[2]Conserved cis-Acting Promoter Elements Are Required for Density-Dependent Transcription of <span style="font-style: italic">Agrobacterium
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tumefaciens</span> Conjugal Transfer Genes, 1995 CLAY FUQUA 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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Latest revision as of 00:14, 2 November 2017

<!DOCTYPE html> Coli Sapiens

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)



Fig. 1 Construction of sample

Negative control (Fig. 2)

C. pSB6A1

D. pSB3K3

Fig. 2 Construction of Negative control

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

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