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Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm
From 2012.igem.org
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1. Construction | 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). | 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). | ||
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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Pluxtet-GFP (JM2.300)…Sample | 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 | 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 | pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plac-GFP (JM2.300)…positive control | ||
2. Strain | 2. Strain | ||
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JM2.300 | JM2.300 | ||
3. Protocol | 3. Protocol | ||
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1.1 Prepare overnight culture of reporter cell at 37 ℃ for 12hours. | 1.1 Prepare overnight culture of reporter cell at 37 ℃ for 12hours. | ||
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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) | 1.2 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) | ||
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1.3 Dilute the flesh culture in 1:50 by the following conditions: | 1.3 Dilute the flesh culture in 1:50 by the following conditions: | ||
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A) LB | A) LB | ||
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B) LB + aTc (500 ng/ ml) | B) LB + aTc (500 ng/ ml) | ||
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C) LB + 3OC6HSL (1 μM ) | C) LB + 3OC6HSL (1 μM ) | ||
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D) LB + aTc (500 ng/ml) + 3OC6HSL (1 μM ) | D) LB + aTc (500 ng/ml) + 3OC6HSL (1 μM ) | ||
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1.4 Incubate the fresh culture of diluted inducer cell for 2 hours. | 1.4 Incubate the fresh culture of diluted inducer cell for 2 hours. | ||
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1.5 Flow cytometer measurements for GFP expression of reporter cell. | 1.5 Flow cytometer measurements for GFP expression of reporter cell. | ||
Revision as of 19:35, 26 September 2012
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.
