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Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm
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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"]] | ||
| - | == | + | ==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]] | ||
| - | + | <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]] | ||
| - | + | <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]] | ||
| - | + | <br><br><br><br><br><br> | |
| - | 2. Strain | + | ==2.Strain== |
JM2.300 | JM2.300 | ||
| - | 3. Protocol | + | ==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
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
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.
