TraI Assay

Introduction

Quorum Sensing is cell-to-cell communication system which is used variety of microorganism. Signal molecular used in Quorum sensing has variety of chemical structure. LuxI is 合成遺伝子 of 3OC6AHL and TraI is 合成遺伝子 of 3OC8AHL. Chemical structures of these molecule are shown Figure. 1. LuxR gene express intracellular LuxR receptor. Signal molecular and this receptor form complex. This complex interacts with responsive promoters, Plux and regulates transcription of downstream genes. The concentration of signal molecular increase with cell density. By using this system, microorganism assess their local density and regulates gene expression.
In previous study, a novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR was developed. In this system, transcription downstream of a region called CMV minimal promoter is induced in the presence of signal molecular 3OC8AHL. Therefore, we chose 3OC8AHL as signal molecule and tried to make E.coli to produce 3OC8AHL.

Summary of experiment

In this section, we confirmed whether E. coli expressing Tral protein truly produced signal molecules, AHL (Acyl Homoserine Lactone).
To achieve this goal, we constructed two types of E. coli. One is the “Sender” E. coli which produces AHL and the other is the “Reporter” E. coli which expresses GFP in the presence of AHL.
To begin with, we evaluated whether “Reporter” cell could express GFP dependent on AHL by culturing them in liquid LB medium containing various concentrations of AHL (0.1nM - 1000nM).
Then we confirmed whether the “Sender” could produce AHL. The supernatant of the “Sender” cell’s medium was added into the medium of “Reporter” cells and the production of AHL was confirmed by the expression of GFP from the “Reporter” cells.
Following plasmids were introduced into E. coli.
Reporter
Fig. 1で示したプラスミドをE.coliのDH5α株にダブルトラフォメすることで、E.coliが3OC8AHLシグナルや3OC6AHLシグナルに反応してGFPを生産するようになる。

Sender
Fig. 2 に示したように、恒常プロモーターであるPtetの下流にTraIをつなぐことによって、常に3OC8AHLを合成するE.coliを作ることができる。

Results

LuxR protein is a receptorfor C6 signals. However, previous study (文献) showed that it can also bind to other kinds of AHL, such as C10 .
We confirmed that LuxR could also respond to C8 signals as sensitive as respoding of C6 signals. Receiver E.coli’s RFU (Reletive Fluoroscent Units) in each AHL concentration (0.01 nM ? 1000 nM) is shown in Figure .
Detection limit was over 10nM in case of C6 and C8. RFU valueswere almost same over 100nM.

Error bar have a same width as standard deviation (n=3).