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Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm
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(1) construction | (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). | + | To characterize Lux-Tet hybrid promoter (BBa-K934024), we constructed Plux/tet-GFP (BBa-K934025) by ligating the Lux-Tet hybrid promoter (BBa-K934024) to the upstream of promoterless GFP generator (BBa-I13504). |
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(3)Strain | (3)Strain | ||
| - | + | JM2.300 | |
(4)protocol | (4)protocol | ||
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Sample: | Sample: | ||
| - | A) pLuxR-Ptrc-GFP ( | + | A) pLuxR-Ptrc-GFP (JM2.300) |
| - | B) pLuxR-ΔP-GFP ( | + | B) pLuxR-ΔP-GFP (JM2.300).... negative control |
| - | C) pLuxR-PLac-GFP ( | + | C) pLuxR-PLac-GFP (JM2.300)… positive control |
Method: | Method: | ||
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1 ,Prepare overnight culture at 37℃ for 12hours. | 1 ,Prepare overnight culture at 37℃ for 12hours. | ||
| - | 2, Take 30μl of the overnight culture of | + | 2, Take 30μl of the overnight culture of samples into LB(3ml) + antibiotics (Amp 50μg/ml).(→fresh culture) |
3,Dilute the flesh culture in 1:50 by the following conditions: | 3,Dilute the flesh culture in 1:50 by the following conditions: | ||
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d) LB + anhydrotetracycline (500ng/ ml) + acylated homoserine lactone(1μM ) | d) LB + anhydrotetracycline (500ng/ ml) + acylated homoserine lactone(1μM ) | ||
| - | 4, Incubate the flesh culture of diluted inducer | + | 4, Incubate the flesh culture of diluted inducer cells for 2 hours. |
5, Flow cytometer measurements for GFP expression of reporter cell. | 5, Flow cytometer measurements for GFP expression of reporter cell. | ||
Revision as of 08:41, 26 September 2012
Materials & Method
[Back to "Lux-Tet hybrid promoter assay"]
(1) construction
To characterize Lux-Tet hybrid promoter (BBa-K934024), we constructed Plux/tet-GFP (BBa-K934025) by ligating the Lux-Tet hybrid promoter (BBa-K934024) to the upstream of promoterless GFP generator (BBa-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
JM2.300
(4)protocol
Sample:
A) pLuxR-Ptrc-GFP (JM2.300)
B) pLuxR-ΔP-GFP (JM2.300).... negative control
C) pLuxR-PLac-GFP (JM2.300)… positive control
Method:
1 ,Prepare overnight culture at 37℃ for 12hours.
2, Take 30μl of the overnight culture of samples into LB(3ml) + antibiotics (Amp 50μg/ml).(→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 cells for 2 hours.
5, Flow cytometer measurements for GFP expression of reporter cell.
