Tokyo/Works/Assay

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Revision as of 19:57, 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

Purpose of this assay (Hill function fitting)

We have obtained data for our newly devised hybrid promoter. By Hill function fitting, we have determined n2, n3, K2, and K3.

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1.AHL assay

Fig.1 The result of AHL assay Fluorescence intensity (arbitrary unit, a.u.) as a function of the concentration of AHL was determined.

Purpose
check how AHL activates lux-lac hybrid promoter.
These parameters(n2,K2) is decided.

Result & Conclusion
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. 1, the characteristics of the hybrid promoter expressed in Hill function, such as (n2,K2), is determined.

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2.IPTG assay

Fig.2 The result of IPTG assay Fluorescence intensity as a function of the concentration of IPTG was determined.

Purpose
check how LacI represses this hybrid promoter. In order to adjust LacI concentration, IPTG was added.
These parameters(n3,K3) is decided.

Result & Conclusion
As the concentration of IPTG increases, GFP fluorescence increased. It indicates that the hybrid promoter in the cells expressing LacI became increasingly strengthened by decline of repression by LacI. The activation graph in Fig. 2, the characteristics of the hybrid promoter expressed in Hill function is determined.

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Appendix: Preliminary assays

We conducted preliminary assays to practice experiments and check if the materials and methods worked properly.

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