TraI Assay

Introduction

Quorum sensing is the cell-to-cell communication system used by a variety of bacteria. Signal molecules used in quorum sensing are chemically diverse, and the acyl-homoserine lactone (AHL)-type molecules are the most studied and employed ones in synthetic biology. luxI (Vibrio fischeri) and traI (Agrobacterium fumigatus) encode the AHL synthases for 3OC6HSL and 3OC8AHL, respectively. Chemical structures of these molecules are shown in Fig. 1.

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The luxR gene of V. fischeri encodes intracellular receptor for 3OC6HSL.The complex of LuxR and 3OC6HSL binds to the responsive promoter, Plux, and activates transcription of downstream genes. Note that the luxI gene is one of such downstream genes. A similar mechanism is present for 3OC8HSL that is produced in A. fumigatus, and in this case, the receptor is encoded by the traR gene. Therefore, for both cases, the positive feedback loop of transcription is formed, and when the concentration of AHLs exceeds a threshold level, specific transcription is induced rapidly. As a consequence, bacterial cells can sense their population density and carry out cell-density specific behaviors such as luminescence emission and pathogenicity exerting.
In a previous study, AHL-inducible eukaryotic gene expression system was developed based on TraR (1). In this system, expression from the eukaryotic promoter (CMV minimal promoter) is induced only in the presence of 3OC8HSL. Therefore, we chose 3OC8HSL as a signal molecule and tried to make E. coli cells produce 3OC8HSL.

Summary of experiment

In this section, we confirmed whether E. coli cells expressing Tral protein produce a practical amount of 3OC8HSL.
To this end, two E. coli strains were constructed; one is the “Sender” strain which produces 3OC8HSL and the other is the “Reporter” strain which expresses GFP in the presence of 3OC8HSL.
To begin with, it was investigated whether the “Reporter” cellscould express GFP depending on 3OC8HSL when cultured in liquid LB medium containing various concentrations of 3OC8HSL (0.1 nM -1000 nM).
In the previous similar experiment, the intensities of GFP fluorescence (Relative Fluorescence Units; RFU) have shown to follow Hill's equation (2). Therfore, in this study, the parameters of Hill's equation were obtained from the data and the concentrations of AHL were calculated from the values of RFU.
Then, whether the “Sender” could produce AHL was investigated. The supernatant of the “Sender” s was added into the actively growing culture of the“Reporter” and the production of AHL was evaluated by observing the expression of GFP.

The following plasmids were introduced into E. coli.
Reporter E.coli
By introducing the plasmids shown in Figure. 2, E. coli cells are expected to produce GFP in response to 3OC8AHL and 3OC6AHL. Note that Ptet is the constitutive promoter. Also, note that LuxR can accept 3OC8AHL as well as the natural ligand, 3OC6AHL (3); we here employed LuxR, but not TraR, because LuxR had been characterized far better than TraR in the preceding iGEM projects.

Sender E.coli
We created the Sender by introducing the plasmid shown in Fig. 3.
The Sender is expected to produce 3OC8AHL constantly, because the traI gene is placed at downstream of the constitutive promoter, Ptet.

Results

Assay using reagent AHLs
In order to analyze the ability of the Reporter to receive AHLs and to express GFP depending on AHL, defined concentrations of reagent AHLs were added to growing culture of the Reporter. It was confirmed that LuxR responded to 3OC8AHL in a similar level to 3OC6HSL. RFU of the Reporter at various AHL concentrations (0.01 nM ? 1000 nM) is shown in Fig. 4. Detection limit was over 10nM for both cases.