Tokyo/AHL assay

From 2007.igem.org

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

Objective of this assay   Effect of AHL   Effect of IPTG　　Preliminary assays

Effect of AHL

Objective:

By testing how AHL activates lux-lac hybrid promoter, parameters(n2,K2) should be determined.

===Samples: === in DH5 alpha
placIQ(constitutive promoter) + GFP (Pos. con.) :[http://partsregistry.org/Part:BBa_J54201 BBa_J54201]
No promoter + GFP (Neg. con.) :[http://partsregistry.org/Part:BBa_J54102 BBa_J54102]
Lux lac hybrid promoter + GFP :[http://partsregistry.org/Part:BBa_I751900 BBa_I751900]

Procedure:

Fig.1: Activated by externally added AHL, cells had accumulated GFP, increasing fluorescence intensity.

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
Fluorescence intensity (arbitrary unit, a.u.) as a function of the concentration of AHL was determined.

The concentration of AHL dose-denpendently increased iGFP fluorescence. This result indicates 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.

n2 = 2.08 (-)
K2 = 4.05 (μM)