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

From 2012.igem.org

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(3.Protocol)
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==Construction ==
==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 Plux/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 Plux/tet hybrid promoter (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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==2.Strain==
==2.Strain==

Revision as of 22:01, 26 September 2012

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Lux-Tet hybrid promoter assay

Materials & Method

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Construction

To characterize Plux/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 Plux/tet hybrid promoter (BBa_K934024) to the upstream of promoterless GFP generator ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I13504 BBa_I13504]).


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

Luxtet13tokyotech.png







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

Luxtet14tokyotech.png







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

Luxtet15tokyotech.png







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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