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

From 2012.igem.org

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[[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Back to "Lux-Tet hybrid promoter assay"]]
[[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Back to "Lux-Tet hybrid promoter assay"]]
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(1) construction
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i. Materials & Methods
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1. Construction
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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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To characterize Lux-Tet hybrid promoter (BBa-K934024), we constructed Plux/tet-GFP (BBa-K934025) by ligating the Lux-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 
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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-⊿P-GFP (JM2.300)…negative control
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(2)Samples
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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plac-GFP (JM2.300)…positive control
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Sample:
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A) pLuxR-Ptrc-GFP (JM2.300)
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B) pLuxR-ΔP-GFP (JM2.300).... negative control
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C) pLuxR-PLac-GFP (JM2.300)… positive control
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(3)Strain
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2. Strain
JM2.300
JM2.300
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(4)protocol
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3. Protocol
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1.1 Prepare overnight culture of reporter cell at 37 ℃ for 12hours.
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Sample:
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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)
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1.3 Dilute the flesh culture in 1:50 by the following conditions:
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A) pLuxR-Ptrc-GFP (JM2.300)
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A) LB
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B) LB + aTc (500 ng/ ml)
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B) pLuxR-ΔP-GFP (JM2.300).... negative control
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C) LB + 3OC6HSL (1 μM )
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D) LB + aTc (500 ng/ml) + 3OC6HSL (1 μM )
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C) pLuxR-PLac-GFP (JM2.300)… positive control
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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.
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Method:
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1 ,Prepare overnight culture at 37℃ for 12hours.  
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2, Take 30μl of the overnight culture of samples into LB(3ml) + antibiotics (Amp 50μg/ml).(→fresh culture)
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3,Dilute the flesh culture in 1:50 by the following conditions:
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a) LB
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b) LB + anhydrotetracycline (500ng/ ml)
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c) LB + acylated homoserine lactone(1μM )
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d) LB + anhydrotetracycline (500ng/ ml) + acylated homoserine lactone(1μM )
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4, Incubate the flesh culture of diluted inducer cells for 2 hours.
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5, Flow cytometer measurements for GFP expression of reporter cell.
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[[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Back to "Lux-Tet hybrid promoter assay"]]
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Revision as of 19:34, 26 September 2012

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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.
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