From 2012.igem.org
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| - | {{tokyotechcss}}
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| - | {{tokyotechtop}}
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| - | {{tokyotechmenubar}}
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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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| - | Lux-Tet hybrid promoter assay </div>
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| - | </div class="whitebox">
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| - | <div class="whitebox">
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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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| - | (1) construction
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| - | To characterize Lux-Tet hybrid promoter (K934024), we constructed Plux/tet-GFP (K934025) by ligating the Lux-Tet hybrid promoter (K934024) to the upstream of promoterless GFP generator (I13504).
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| - | (2)Samples
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| - | Sample:
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| - | A) pLuxR-Ptrc-GFP (JM2300)
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| - | B) pLuxR-ΔP-GFP (JM2300).... negative control
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| - | C) pLuxR-PLac-GFP (JM2300)… positive control
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| - | (3)Strain
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| - | JM2300
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| - | (4)protocol
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| - | Sample:
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| - | A) pLuxR-Ptrc-GFP (JM2300)
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| - | B) pLuxR-ΔP-GFP (JM2300).... negative control
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| - | C) pLuxR-PLac-GFP (JM2300)… positive control
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| - | Method:
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| - | 1 ,Prepare overnight culture at 37℃ for 12hours.
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| - | 2, Take 30μl of the overnight culture of sample into LB(3ml) + antibiotics (Amp 6μl).(→fresh culture)
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| - | 3,Dilute the flesh culture in 1:50 by the following conditions:
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| - | a) LB
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| - | b) LB + anhydrotetracycline (500ng/ ml)
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| - | c) LB + acylated homoserine lactone(1μM )
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| - | d) LB + anhydrotetracycline (500ng/ ml) + acylated homoserine lactone(1μM )
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| - | 4, Incubate the flesh culture of diluted inducer cell for 2 hours.
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| - | 5, Flow cytometer measurements for GFP expression of reporter cell.
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Latest revision as of 00:04, 27 October 2012
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