Team:TokyoTech/Experiment/TraI Assay

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iGEM Tokyo Tech

TraI Assay

Overview

In this section we confirmed that E.coli containing Trail genes (we call it Seder E.coli) truly produce signal molecule, AHL. At first to detect small amount of AHL we made reporter E.coli which respond to AHL and produce GFP. Then we studied AHL concentration dependence of reporter’s fluorescent intensity. At last, we mix supernatant of Sender E.coli media with culture in which reporter grow and measure intensity of fluorescence and confirmed E.coli produce AHL (Acyl Homoserine Lactone).

Results

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LuxR is Receptor gene for C6 signals. But it is known LuxR bind to other AHL such as C10 and cause crosstalk. We confirmed LuxR respond to C8 signals and have almost same sensitibvity in the case of C6 signals. Receiver E.coli’s RFU (Reletive Fluoroscent Units) in each concentration (1µM,100nM,10nM…) is shown in Figure . Error bar have a same width as standard deviation (n=3). Detection limit is over 10nM in case of C6 and C8. RFU values are almost same over 100nM.

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Supernatant Assay

Supernatant Assay Strain dependence of AHL production We found that Amount of C8 production is depend on E.coli’s strain. RFU is ○○ folds larger than DH5α.

Supernatant Assay

Temperature dependence of AHL production. We also found that Amount of C8 production is depend on culture temperature. RFU is ○○ folds larger than DH5α.

Discussion

考察

Reference

参考文献

Hajime Fujita: All Rights Reserved