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. luxI (Vibrio fischeri) and traI (Agrobacterium fumigatus) encode the AHL synthases for 3OC6HSL (C6) and 3OC8HSL (C8), respectively. Chemical structures of these molecules are shown in Fig. 1.

The luxR gene of V. fischeri encodes intracellular receptor for C6. The complex of LuxR and C6 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 C8, 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, an 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 C8. Therefore, we here chose C8 as a signal molecule and tried to engineer E. coli cells to produce C8.

Summary of experiment

In this section, we investigated whether E. coli cells expressing TraI protein produce a practical amount of C8.
To this end, two E. coli strains were constructed; one is the “Sender” strain which produces C8 and the other is the “Reporter” strain which expresses GFP in the presence of C8.
To begin with, it was investigated whether the “Reporter” cells expressed GFP when cultured in the medium containing various concentrations of C8 (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). Therefore, 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, the ability of the Sender to produce AHL was analyzed. The supernatant of the Sender was mixed with 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 Fig. 2, E. coli cells are expected to produce GFP in response to C8 and C6. Note that Ptet is the constitutive promoter. Also, note that LuxR can accept C8 as well as the natural ligand, C6 (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 C8 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 C8 in a similar level to C6. 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 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.

Supernatant Assay
Temperature dependence of AHL production.
During the trial-and-error for increasing the productivity of AHL in the Sender, we found that the amount of C8 was higher when the Sender was grown at a lower temperature; the RFU value was 14-fold higher.
E. coli introduced empty vector was used as Negative Control.
C8 concentrations were calculated from the RFU values using calibration curve, 34 nM of C8 was produced in the 37℃ culture, whereas C8 concentration exceeded the detection limit in the 25℃ culture.

Discussion

We confirmed that E. coli produces over 200 nM of C8. 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 improve the production amount of C8 further.
The result in Fig. 5 shows that temperature dependency of C8 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 (5).

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). Hilde Hansen, Amit Anand Purohit, Hanna-Kirsti S Leiros, Jostein A Johansen, Stefanie J Kellermann, (2015) The autoinducer synthases LuxI and AinS are responsible for temperature-dependent AHL production in the fish pathogen Aliivibrio salmonicida Ane Mohn Bjelland2 and Nils Peder Willassen1, Hansen et al. BMC Microbiology (2015) 15:69
(5). Elise R. Morton and Clay Fuqua (2012) UNIT 3D.1 Laboratory Maintenance of Agrobacterium. Curr Protoc Microbiol. 2012 Feb; CHAPTER: Unit3D.1.