Materials & Method

[Back to "Lux-Tet hybrid promoter assay"]

Construction

To characterize Plux/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 Plux/tet hybrid promoter (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-Pluxtet-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 ℃ 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.