Lux-Tet hybrid promoter assay

Materials & Method

(1) construction

To characterize Lux-Tet hybrid promoter (K934024), we constructed Plux/tet-GFP (K934025) by ligating the Lux-Tet hybrid promoter (K934024) to the upstream of promoterless GFP generator (I13504).

(2)Samples

Sample:

A) pLuxR-Ptrc-GFP (JM2300)

B) pLuxR-ΔP-GFP (JM2300).... negative control

C) pLuxR-PLac-GFP (JM2300)… positive control

(3)Strain

JM2300

(4)protocol

Sample:

A) pLuxR-Ptrc-GFP (JM2300)

B) pLuxR-ΔP-GFP (JM2300).... negative control

C) pLuxR-PLac-GFP (JM2300)… positive control

Method:

1 ,Prepare overnight culture at 37℃ for 12hours.

2, Take 30μl of the overnight culture of sample into LB(3ml) + antibiotics (Amp 6μl).(→fresh culture)

3,Dilute the flesh culture in 1:50 by the following conditions:

a) LB

b) LB + anhydrotetracycline (500ng/ ml)

c) LB + acylated homoserine lactone(1μM )

d) LB + anhydrotetracycline (500ng/ ml) + acylated homoserine lactone(1μM )

4, Incubate the flesh culture of diluted inducer cell for 2 hours.

5, Flow cytometer measurements for GFP expression of reporter cell.