Tokyo/AHL assay

From 2007.igem.org

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(Procedure:)
(Procedure:)
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<br>The final concentration of AHL (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM  
<br>The final concentration of AHL (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM  
<br>(*The same amount of DMSO, solvent for AHL, was added to each sample.)
<br>(*The same amount of DMSO, solvent for AHL, was added to each sample.)
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<br>incubate for 2 to 3 hours  
<br>incubate for 2 to 3 hours  
<br>apply 150 ul of samples into 96-well plaste  
<br>apply 150 ul of samples into 96-well plaste  

Revision as of 11:03, 25 October 2007


Works top  0.Hybrid promoter  1.Formulation  2.Assay1  3.Simulation  4.Assay2  5.Future works

Purpose of this assay  1.AHL assay  2.IPTG assay  Preliminary assays

AHL assay

Purpose:


To check how AHL activates the newly devised lux-lac hybrid promoter

Samples:

Pos. con. Neg. con. Lux lac hybrid promoter


Procedure:

Fig.1: Externally added AHL


prepare overnight culture for each sample
make fresh culture
take 3 ul of the overinight culture into 3 ml of LB (+ Amp and 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
The final concentration of AHL (in 3 ml LB culture) = 0, 500, 1000, 2000, 4000, 5000, 5500, 6000, 6500, 7000, 8000, 9000, and 10000 nM
(*The same amount of DMSO, solvent for AHL, was added to each sample.)
incubate for 2 to 3 hours
apply 150 ul of samples into 96-well plaste
FLA measurement

Result & Conclusion:

Fig.2: Result of AHL assay


As the concentration of AHL increases, GFP fluorescence increased, indicating that the hybrid promoter’s activation is strengthend with increasing concentration of AHL. From the activation graph in Fig. 2, the characteristics of the hybrid promoter expressed in Hill function, such as (n2,K2), is determined.