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Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm
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Band detect system assay </div> | Band detect system assay </div> | ||
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Revision as of 08:58, 11 October 2012
Materials & Method
[Back to "Band detect system"]
Construction
To characterize lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]), we constructed Plux/tet-GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934025 BBa_K934025]) by ligating the lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]) to the upstream of promoterless GFP generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I13504 BBa_I13504]).
pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plux/tet-GFP (JM2.300)…Sample
pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-⊿P-GFP (JM2.300)…negative control
pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plac-GFP (JM2.300)…positive control
2.Strain
JM2.300
3.Protocol
1.1 Prepare overnight culture of reporter cell at 37°C for 12hours.
1.2 Take 30 μl of the overnight culture of reporter cells into LB (3 ml) + antibiotics (Amp 50 μg/ml + Kan 30 μg/ml). (→fresh culture)
1.3 Dilute the flesh culture in 1:50 by the following conditions:
A) LB
B) LB + aTc (500 ng/ ml)
C) LB + 3OC6HSL (1 μM )
D) LB + aTc (500 ng/ml) + 3OC6HSL (1 μM )
1.4 Incubate the fresh culture of diluted inducer cell for 2 hours.
1.5 Flow cytometer measurements for GFP expression of reporter cell.
[Back to "Lux-Tet hybrid promoter assay"]
