Tokyo/AHL assay
From 2007.igem.org
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<br>incubate for 2 to 3 hours until the observed OD is around 1.2 (Falcon tube = 14 mm in daimeter) | <br>incubate for 2 to 3 hours until the observed OD is around 1.2 (Falcon tube = 14 mm in daimeter) | ||
<br>add AHL & IPTG solution | <br>add AHL & IPTG solution | ||
| - | <br>[AHL]final (in 3 ml LB culture) = | + | <br>[AHL]final (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM |
<br>[IPTG]final (in 3 ml LB culture) = 1 mM | <br>[IPTG]final (in 3 ml LB culture) = 1 mM | ||
<br>incubate for 2 to 3 hours | <br>incubate for 2 to 3 hours | ||
Revision as of 05:42, 25 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) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 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.
