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

From 2012.igem.org

(Difference between revisions)
(1. Construction)
(1. Construction)
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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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==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   
[[File:luxtet13tokyotech.png|200px|left]]
[[File:luxtet13tokyotech.png|200px|left]]
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<br><br><br><br><br><br>
pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-⊿P-GFP (JM2.300)…negative control
pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-⊿P-GFP (JM2.300)…negative control
[[File:luxtet14tokyotech.png|200px|left]]
[[File:luxtet14tokyotech.png|200px|left]]
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<br><br><br><br><br><br>
pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plac-GFP (JM2.300)…positive control
pSB3K3-PlacIq-LuxR-Ptrc-TetR / pSB6A1-Plac-GFP (JM2.300)…positive control
[[File:luxtet15tokyotech.png|200px|left]]
[[File:luxtet15tokyotech.png|200px|left]]
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<br><br><br><br><br><br>
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2. Strain
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==2.Strain==
JM2.300
JM2.300
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3. Protocol
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==3.Protocol==
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
<div id="tokyotechprotocol" style=" font:bold ;left ; font-size: 13px; color: #000000; padding: 10px;">
1.1 Prepare overnight culture of reporter cell at 37 ℃ for 12hours.
1.1 Prepare overnight culture of reporter cell at 37 ℃ for 12hours.

Revision as of 19:43, 26 September 2012

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Lux-Tet hybrid promoter assay

Materials & Method

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

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

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.

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