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

From 2012.igem.org

Revision as of 19:34, 26 September 2012 by Takuo (Talk | contribs)

bar

Tokyotechlogo2012.png

Lux-Tet hybrid promoter assay

Materials & Method

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

i. Materials & Methods 1. 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 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.