Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm

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Lux-Tet hybrid promoter assay

Materials & Method

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Construction

To characterize Plux/tet hybrid promoter (BBa_K934024), we constructed Plux/tet-GFP (BBa_K934025) by ligating the Plux/tet hybrid promoter (BBa_K934024) to the upstream of promoterless GFP generator (BBa_I13504).


pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Pluxtet-GFP (JM2.300)…Sample

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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-⊿P-GFP (JM2.300)…negative control

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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plac-GFP (JM2.300)…positive control

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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.

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