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.

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 10 nM for both cases.

The data are presented as mean ± SD from triplicate experiments.

Based on the data which is shown in Fig. 4, parameter was obtained to fit Hill’s equation.
Hill’s equation is shown in Eq. 1

The values of parameters are shown in Table. 1
The parameter “a” represents leakiness of the GFP expression in the Receiver. Even in the absence of AHL, it is known that downstream genes below Plux are transcribed slightly. The parameter “b” is the value of RFU when AHL binds to all receptors and is completely induced. The parameter “n“ is the Hill coefficient, and when this value is 1 or more, it is said that there are multiple binding sites. “Km” is the AHL concentration where half of the receptor molecules is bound to the AHL molecules, and this value represent the detection sensitivity of the Reporter. It was found that both AHLs can be detected with a sensitivity of order 10 nM.

The actual measurement value and the theoretical formulaare shown in Fig. 5. In both graphs, the RFU increased greatly over the range between 10 and 100 nM, and it was found that more than 100 nM of AHL, can not be directly quantified.

Supernatant Assay
Temperature dependence of AHL production.
During the trial-and-error to increase the productivity of AHL in the Sender, we found that the amount of C8 produced is dependent on the culture temperature of the Sender. RFU was 14 folds larger than DH5α.
3OC8HSL concentration of TraI culture in 37℃ was 37 nM.　The RFU values of mixture in which Sender E. coli was cultivated in25℃ exceeded the detection limit.

Discussion

We confirmed that E. coli produces over 200 nM of 3OC8AHL. However, as shown in other wiki pages, the final objective of our project is inducing gene expression with C8 in mammalian cells. The previous study (1) showed that at least1 μM of C8 was required to do so. Therefore, we need to improvemeC8 production further.
The result in Figure 5 shows that temperature dependency of 3OC8AHL production. This result may reflect that the traI gene is derived from a soil bacterium A. tumefaciens in nature, the temperature of soil hardly reaches 37 ℃, and the TraI protein may be unstable at 37℃. Indeed, growth of A. tumefaciens occurs optimally at 28°C, and at temperatures above 30°C, A. tumefaciens becomes heat-shock state (4).

Appendix: Material and Method

Reference

(1). Neddermann P1, Gargioli C, Muraglia E, Sambucini S, Bonelli F, De Francesco R, Cortese R (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR. EMBO Rep. 2003 Feb;4(2):159-65.
(2). https://2014.igem.org/Team:ETH_Zurich/modeling/qs
(3). https://2016.igem.org/Team:Tokyo_Tech/AHL_Assay/AHL_Reporter_Assay
(4). Elise R. Morton and Clay Fuqua* (2012) UNIT 3D.1 Laboratory Maintenance of Agrobacterium. Curr Protoc Microbiol. 2012 Feb; CHAPTER: Unit3D.1.