Difference between revisions of "Team:TokyoTech/Experiment/TraR Reporter Assay"
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<p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">- Plasmids</p> | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">- Plasmids</p> | ||
<p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">Sample(Fig. 1)</p> | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">Sample(Fig. 1)</p> | ||
| − | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">A. Ptet – rbs – traR (pSB6A1)</p> | + | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">A. Ptet – <span style="font-style: italic">rbs – traR</span> (pSB6A1)</p> |
| − | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">B. Ptra – rbs – gfp (pSB3K3) </p><br></br> | + | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">B. Ptra – <span style="font-style: italic">rbs – gfp</span> (pSB3K3) </p><br></br> |
<p style="font-family: Poppins;font-size: 16px"><p style="text-indent: | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent: | ||
<figure> | <figure> | ||
| − | <img src="" style="max-width:60%"> | + | <img src="https://static.igem.org/mediawiki/2017/9/9d/T--TokyoTech--Plasmid_sample.png" style="max-width:60%"> |
| − | <figcaption style="font-family: Poppins;font-size: 16px">Fig. 1 Ptet - rbs - traR - tt (pSB6A1) Ptra - rbs - gfp - tt (pSB3K3)</figcaption> | + | <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> |
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<p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">D. pSB3K3 </p> | <p style="font-family: Poppins;font-size: 16px"><p style="text-indent:1em">D. pSB3K3 </p> | ||
<figure> | <figure> | ||
| − | <img src="" style="max-width:60%"> | + | <img src="file:///Users/hasegawahazuki/Desktop/iGEM17/iGEM17Wiki/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 pSB6A1 pSB3K3</figcaption> | ||
</figure> | </figure> | ||
Revision as of 15:44, 29 October 2017
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TraR Reporter Assay
Introduction
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 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)
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.
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
