Difference between revisions of "Part:BBa K2505010"

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Ptet-rbs-traI-tt

Sequence and Features

This part constitutively produces C8.

Characterization

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. 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.

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.

Fig.2 Structure of the plasmids used for creating the “Reporter”

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.

Result

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.

Fig.4 Concentration dependance of Reletive Fluoroscent Units 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

Eq.1 Hill's equation 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. Table. 1 Parameters of Hill’s equation

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.

Fig. 5 Actual measurement value and Theoretical formula

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 34 nM.　The RFU values of mixture in which Sender E. coli was cultivated in25℃ exceeded the detection limit.

Fig.6 Temperature dependency of 3OC8HSL production

Material and Method

Materials

Strains Reporter ・DH5α Sender E.coli ・DH5α Medium ・LB medium ・LB medium containing antibiotics Dissolve antibiotics in LB medium (Ampicillin 50μg/mL, Chloramphenicol 34μg/mL, Kanamycin 50μg/mL)

Methods

Reagent assay 1. Grow the Receiver in LB medium containing appropriate antibiotics for about 15 hours. 2. Dilute the culture to 1/200 with flesh LB medium containing antibiotics 3. Incubate the fresh culture for 2 hours 4. Mix 495 μL of the culture with 5 μL of AHL solution in a microtube (each AHL solution was dissolved in DMSO and contained 100 microM, 10 microM...of AHL to give a final concentration of 1 microM 100 nM..., respectively) 5. Incubate the microtube for 5 hours with gentle shaking at 37℃ 6. Take 100 μL of culture and measure fluorescence intensity (excitation and emission wave lengths are 495 and 520 nm, respectively) and turbidity (measurement wavelength is 600nm) 7. Calculate RFU dividing fluorescence intensity by turbidity and minus control value. Supernatant Assay 1. Grow the Sender in LB medium for about 15 hours at each temperature (37℃, 25℃). 2. Harvest the cells by brief centrifugation 3. Perform the same procedure as the reagent assay process (1~4) to prepare Reporter culture. 4. Mix 250 μL of the supernatant of Sender culture with Reporter culture in a microtube. 5. Incubate the microtube for 5 hours with gentle shakingat 37℃ 6. Take 100 μL of the culture and measure fluorescence intensity (excitation and emission wave lengths are 495 and 520 nm, respectively) and turbidity (measurement wavelength is 600 nm) 7. Calculate RFU dividing fluorescence intensity by turbidity and minus control value.