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

From 2012.igem.org

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Band detect system assay </div>
 
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__NOTOC__
 
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=Materials & Method=
 
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[[http://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Go to the project page "Band detect system"]]
 
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==Construction ==
 
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To characterize lux/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 lux/tet hybrid promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934024 BBa_K934024]) to the upstream of promoterless GFP generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I13504 BBa_I13504]).
 
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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plux/tet-GFP (JM2.300)…Sample 
 
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[[File:luxtet13tokyotech.png|400px|center]]
 
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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-⊿P-GFP (JM2.300)…negative control
 
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[[File:luxtet14tokyotech.png|400px|center]]
 
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pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plac-GFP (JM2.300)…positive control
 
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[[File:luxtet15tokyotech.png|400px|center]]
 
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==2.Strain==
 
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JM2.300
 
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==3.Protocol==
 
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1.1 Prepare overnight culture of reporter cell at 37°C 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)
 
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1.3 Dilute the flesh culture in 1:50 by the following conditions:
 
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A) LB
 
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B) LB + aTc (500 ng/ ml)
 
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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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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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[[http://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Go to the project page "Lux-Tet hybrid promoter assay"]]
 

Latest revision as of 00:04, 27 October 2012