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Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm
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Revision as of 08:54, 25 September 2012
Materials & Method
[Back to "Lux-Tet hybrid promoter assay"]
(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.
