TraI Assay

Introduction

In this section we confirmed that E.coli containing Trail genes (we call it Seder E.coli) truly produce signal molecule, AHL(Acyl Homoserine Lactone). 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.

Summary of experiment

We use following plasmids and induced into E.coli. LuxR and Plux is Lux system Receptor and Promorter.

Results

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.

Supernatant Assay

Temperature dependence of AHL production.

We found that Amount of C8 production is depend on culture temperature. RFU is 14 folds larger than DH5α.

Discussion

To send a molecular signal AHL to mammalian cells, our goal is 20μM of C8 production. Receiver E.coli’s sensitivity is enough to detect such a small amount of AHL. But Sender E.coli’s C8 production is not enough to send AHL. TraI is derived from soil microorganism A. Tumefaciens. It is rarely happen that Temperature of the soil rise above 37 ℃. Therefore It is considered that TraI gene does not work properly above 37℃ of culture temperature.

Reference

参考文献