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Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm
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__NOTOC__ | __NOTOC__ | ||
| - | = | + | =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. | ||
Revision as of 06:48, 25 September 2012
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.
