Part:BBa_K1139110:Experience

Pcon-LasR-Plux/tet-GFP

Materials & Methods

   1. Overnight Culture -> Fresh Culture -> Induction

1.1 Prepare overnight culture of each cell (GFP posi, GFP nega, sample) at 37°C for 12 h.
(=> O/N)

1.2 Take 30 µL (from GFP posi, GFP nega, sample) of the overnight culture of inducer cell into
LB (3 mL) + antibiotics (Amp 50 µg/mL+ Kan 30 µg/mL).
(=> Fresh Culture)

1.3 Incubate the flesh culture of cells (GFP posi, GFP nega, sample) until the observed OD600 reaches around 0.50.

1.4a Take 30 µL each cell suspensions (GFP posi , GFP nega , sample) into

LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL)
+ 5 µM 3OC6HSL (3 µL),

LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL)
+ 5 µM 3OC6HSL (3 µL) + 0.05 µg/mL aTc (3 µL),

LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL)
+ 5 µM 3OC12HSL (3 µL),

LB (3 mL) + antibiotics (Amp 50 µg/mlL+ Kan 30 µg/mL)
+ 5 µM 3OC12HSL (3 µL)+ 0.05 mg/mL aTc (3 µL),

LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL)
+ 5 µM DMSO (3 µL),

LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL)
+ 5 µM DMSO (3 µL) + 0.05 mg/mL aTc (3 µL).

1.4b Take 30 µL each cell suspensions (GFP posi , GFP

nega , sample) into

LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL)
+ C6 (3 µL) + aTc (3 µL) … inducer concentration for each sample is shown in below (Fig 3-2-3)

	aTc(µg/ml)
C6(nM)		0	0.05	0.15	0.5	1.5	5
	0	No. 1	No. 2	No. 3	No. 4	No. 5	No. 6
	0.3	No. 7	No. 8	No. 9	No. 10	No. 11	No. 12
	1	No. 13	No. 14	No. 15	No. 16	No. 17	No. 18
	3	No. 19	No. 20	No. 21	No. 22	No. 23	No. 24
	10	No. 25	No. 26	No. 27	No. 28	No. 29	No. 30
	30	No. 31	No. 32	No. 33	No. 34	No. 35	No. 36
	100	No. 37	No. 38	No. 39	No. 40	No. 41	No. 42

Fig. 3-2-3. Inducer concentration for each sample

1.5 Incubate all samples (6 samples X 6 kinds of culture = 36 samples) for another 4 h at 37°C.
(=> Induction)

   2. Measurement (Flow cytometer)

2.1 Measure all samples' OD600.

2.2 Dilute all samples with 1X PBS to keep OD600 in the range from 0.2 to 0.5.

2.3 Take 1 mL (from all samples) into disposable tube (for flow cytometer).

2.4 Centrifuge them at 9,000 g, 4°C, 1 min. and take their supernatant away.

2.5 Suspend all samples with 1 mL 1X PBS.

2.6 Measure all samples.

2.7 Save and organize data.

Result

In the graph below (Fig. 1), the level of GFP expression in cells where TetR is active is clearly lower than when TetR is inhibited. This fact could be confirmed in bins of 3OC12HSL and C3OC6HSL. In short, the graph below shows that lux/tet hybrid promoter is repressed by TetR precisely. Furthermore, the graph below shows that there is a great difference between GFP fluorescence intensity of 3OC6HSL + aTc and that of 3OC12HSL + aTc. Now, therefore, crosstalk circumvention experiment is successful.

Fig. 1. The result of crosstalk circumvention

For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/Crosstalk_Circumvention_Assay our work in Tokyo_Tech 2013 wiki].

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