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

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(Materials & Method)
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Lux-Tet hybrid promoter assay </div>
 
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__NOTOC__
 
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=Materials & Method=
 
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[[https://2012.igem.org/Team:Tokyo_Tech/Project#Construction_of_the_positive_feedback_system Back to "Construction of the positive feedback system"]]
 
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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.
 

Latest revision as of 00:04, 27 October 2012