Tokyo/AHL assay

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(Difference between revisions)
(Samples:)
(Purpose:)
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===Purpose: ===
===Purpose: ===
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<br>To check how AHL activates lux-lac hybrid promoter  
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<br>To check how AHL activates the newly devised lux-lac hybrid promoter  
<!--<br>check how lacI represses lux-lac hybrid promoter-->
<!--<br>check how lacI represses lux-lac hybrid promoter-->

Revision as of 06:00, 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:


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

Samples:

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/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:

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.