Tokyo/AHL assay
From 2007.igem.org
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__NOTOC__ | __NOTOC__ | ||
| + | ==[[Tokyo_Tech|Abstract]] [[Tokyo/Model|Concept & Model]] [[Tokyo/Requirements |Requirements]] [[Tokyo/Genetic circuit|Genetic_circuit]] [[Tokyo/Works|Works]] [[Tokyo/About our team|About_our_team]]== | ||
| + | <br>[[Tokyo/Works|Works top]] 0.[[Tokyo/Works/Hybrid promoter|Hybrid promoter]] 1.[[Tokyo/Works/Formulation |Formulation]] 2.[[Tokyo/Works/Assay |Assay1]] 3.[[Tokyo/Works/Simulation |Simulation]] 4.[[Tokyo/Works/Assay2 |Assay2]] 5.[[Tokyo/Works/Future works |Future works]] | ||
== AHL assay == | == AHL assay == | ||
Revision as of 21:43, 24 October 2007
Abstract Concept & Model Requirements Genetic_circuit Works About_our_team
Works top 0.Hybrid promoter 1.Formulation 2.Assay1 3.Simulation 4.Assay2 5.Future works
AHL assay
Purpose:
check how AHL activates lux-lac hybrid promoter
check how lacI represses lux-lac hybrid promoter
Samples:
hybrid promoter plasmid with pTrc99A
hybrod promoter plasmid with pBR322
luxR plasmid with pTrc99A
luxR --- AHL-dependent activation confirmed
placIq on promter-less GFP in DH5a (for pos. con.)
promoter-less GFP in DH5a (for neg. con.)
Procedure:
prepare overnight culture for each sample
make fresh culture
take 3 ul of the overinight culture into 3 ml of LB (Amp and/or Kan) in Falcon tubes.
incubate for 2 to 3 hours until the observed OD is around 1.2 (Falcon tube = 14 mm in daimeter)
add AHL & IPTG solution
[AHL]final (in 3 ml LB culture) = 10 nM
[IPTG]final (in 3 ml LB culture) = 1 mM
incubate for 2 to 3 hours
apply 150 ul of samples into 96-well plaste
FLA measurement
Result & Conclusion:
AND gate by AHL & IPTG
Lux-lac hybrid promoter is activated only in the presence of AHL and IPTG.
