From 2012.igem.org
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| - | {{tokyotechcss}}
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| - | <div class="whitebox">
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| - | <div id="tokyotech" style=" font:bold ;left ; font-size: 50px; color: #1E90FF; padding: 10px;">
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| - | Band detect system assay </div>
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| - | {{tokyotechmenubar}}
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| - | <div id="tokyotech" style=" font:Arial ;left ; font-size: 15px; color: #000000; padding: 30px;">
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| - | __NOTOC__
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| - | =Materials & Method=
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| - | [[https://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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| - | [[https://2012.igem.org/Team:Tokyo_Tech/Project#Band_detect_system Go to the project page "Lux-Tet hybrid promoter assay"]]
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Latest revision as of 00:04, 27 October 2012
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