We confirmed the appearance of the positive feedback from the co-culture assay above. To further confirm the positive feedback system, we characterized the time-dependent change of this positive feedback in the cell-cell communication. To observe time-dependent change of the positive feedback, we prepared four conditions of co-culture : Plux-LasI cell and Plas-LuxI cell coexisted in condition i, ΔP-LasI cell and Plas-LuxI cell in condition ii, Plux-LasI cell and ΔP-LuxI cell in condition iii, and ΔP-LasI cell and ΔP-LuxI cell in condition iv. For a trigger of the positive feedback system, we added the initial dose of 3OC6HSL (5 nM) to each co-culture.

Fig2-1-3-1-11,Four conditions prepared for Time-dependent change assay

To estimate the concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback, the signals content in the supernatants of the co-cultures were collected at the time of 0, 0.5, 1, 2, 4 hours. The concentration of 3OC6HSL and 3OC12HSL produced by the positive feedback was evaluated by Las reporter strain and Lux reporter strain (Fig2-1-3-1-11). Fig2-1-3-1-11 also shows ideal signal production of the positive feedback in the cell-cell communication.

Fig2-1-3-1-12,Time-dependent change assay

Fig2-1-3-1-12 shows the characterization of time-dependent signal increase in the positive feedback in the cell-cell communication. Solid lines represent the fluorescence intensities of Las reporters in the four conditions. Dotted lines represent the fluorescence intensities of Lux reporters in the four conditions.

As compared red solid line with blue dotted line in the condition i (both Plux-LasI cell and Plas-LuxI cell coexist), the fig shows that the fluorescence intensity of Las reporter increases at first (0-1h), and then that of Lux reporter starts to increase (1-2h). This result indicates that the 3OC12HSL production in Plux-LasI cell was activated by initially added 3OC6HSL, whereas the 3OC6HSL production in Plas-LuxI cell was not activated till 3OC12HSL production in Plux-LasI cell reached sufficient level. This behavior strongly suggests the appearance of the positive feedback.

Fig2-1-3-1-12 also suggests that the advantage in signal production in the presence of both signal producers, Plux-LasI cell and Plas-LuxI cell. Only when Plux-LasI cell and Plas-LuxI cell were cultured together and allowed to communicate with one another, the fluorescence intensity of Las reporter and Lux reporter increased drastically. On the other hand, when either Plux-LasI cell or Plas-LuxI cell existed (condition ii and iii), drastic rearing in the GFP expression was not observed. Especially in the condition iii, GFP in the las reporter cell is expressed to some extent (about 2-folds lower than in condition i). This behavior shows that the production of 3OC12HSL in the condition iii cannot exceed the production of 3OC12HSL in the positive feedback state. This is because ΔP-LuxI cell cannot produce any 3OC6HSL to activate Plux-LasI cell in this condition, though initially added 3OC6HSL activated Plux-LasI cell and Plux-LasI cell produced 3OC12HSL to some extent. Moreover, it goes without saying that, the fluorescence intensity of both Las reporter and Lux reporter remained low level through observation in the absence of both signal producer cell.

In addition, low concentration of 3OC6HSL was detected by the Lux reporter in the condition ii, iii & iv, in which 3OC6HSL is not produced. However, fluorescence intensity in these conditions decreased with time. Therefore, it is indicated that these GFP expressions were derived from initially added 3OC6HSL.

From these overall results, we further confirmed the positive feedback in the cell-cell communication and characterized time-dependent change of the positive feedback. Only when the two types of signal producer cells were cultured together and allowed to communicate with one another, signal content in the supernatant of the co-culture drastically increased as compared with other conditions.

Fig2-1-3-1-13,Time-dependent change assay(t=0)

Fig2-1-3-1-14,Time-dependent change assay(t=0.5)

Fig2-1-3-1-15,Time-dependent change assay(t=1)

Fig2-1-3-1-16,Time-dependent change assay(t=2)

Fig2-1-3-1-17,Time-dependent change assay(t=4)

Materials & Methods

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1,Construction

pSB6A1-Ptet-LuxR / pSB3K3-Plux-LasI (JM2.300)…Plux-LasI cell

pSB6A1-Ptrc-LasR / pSB3K3-Plas-LuxI (JM2.300)…Plas-LuxI cell

pSB6A1-Ptet-LuxR / pSB3K3-ΔP-LasI (JM2.300)…ΔP-LasI cell

pSB6A1-Ptrc-LasR / pSB3K3-ΔP-LuxI (JM2.300)…ΔP-LuxI cell

pSB6A1-Ptrc-LasR / pSB3K3-Plas-GFP (JM2.300)…Las reporter cell

pSB6A1-Ptet-LuxR / pSB3K3-Plux-GFP (JM2.300)…Lux reporter cell

pSB6A1-Ptrc-LasR / pSB3K3-ΔP-GFP (JM2.300)…negative control

pSB6A1-Ptrc-LasR / pSB3K3-pλ-GFP (JM2.300)…positive control

2,Strain

JM2.300

Protocol

1. Collect liquid culture

1.1 Prepare overnight culture of inducer cells at 37 °C for 12 hours.

1.2 Take 30 μl of the overnight culture of inducer cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture)

1.3 Incubate the flesh culture of inducer cells until the observed OD600 reaches around 0.50. Centrifuge the cells at 5000 g, 25 °C, 1 min, suspend it with 1 ml LB + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml).

1.4 According to the Fig, take 30 μl cell suspensions into LB(3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml) + 3OC6HSL (5 nM).

Fig2-1-3-1-11,Four conditions prepared for Time-dependent change assay

1.5 Incubate the inducer cells at 37 °C.

1.6 After incubating 0 ,0.5 ,1 ,2 ,4 hours, centrifuge each inducer cell at 9000 g, 4 °C, 1 min, and filter these cultured cells.

1.7 Samples are moved from round tube to 1.5ml tube and frozen in liquid nitrogen.

1.8 Preserve the samples at -80 °C.

1.9 Dissolve these samples at 37 °C just before process 2.5 ,and dilute the filtrate by LB + antibiotics (Amp + Kan) in 1:30.

2 Reporter assay