Tokyo/IPTG assay

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Revision as of 20:56, 26 October 2007

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 LacI-IPTG　　Preliminary assays

LacI-IPTG assay

Objective:

To determine the order of the concentration of IPTG necessary for the activation of our lux-lac hybrid promoter in the LacI producing pTrc99A cells.
The order of the concentration is used for more detailed assay with narrower range of the IPTG concentration.

Samples:

A4 pcI(BBa_I751103) into pTrc99A cell
A4 ΔP(BBa_I751100) into pTrc99A cell
A4 ΔP + Lux-lac hybrid promter (BBa_I751101) into pBR322 cell
A4 ΔP + Lux-lac hybrid promter (BBa_I751101) into pTrc99A cell

Procedure:

Fig.1: As more IPTG is exogenously (externally) added, the fluorescence becomes more intense in the presence of 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 0.5
add AHL & IPTG solution
[AHL]final (in 3 ml LB culture) = 10 nM
[IPTG]final (in 3 ml LB culture) = 1000, 100, 10, 1, 0.1, 0.01, and 0 mM
incubate for 2 to 3 hours
apply 150 ul of samples into 96-well plaste
FLA measurement

Result & Conclusion:

Fig.2: Fluorescence intensity as a function of the concentration of IPTG was determined.

As the concentration of IPTG increases, GFP fluorescence increased, indicating that the hybrid promoter in the LacI expressing cell became increasingly strengthened by decreasing repression by LacI. The activation graph in Fig. 2, the characteristics of the hybrid promoter expressed in Hill function is determined.

n3 = 2.47 (-)
K3 = 0.295 (μM)