Tokyo/Works/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 LacI-IPTG　　Preliminary assays

Objective 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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Effect of AHL

Fig. 1 Effect of AHL on hybrid promoter Enhanced fluorescence intensity represents up-regulation of LuxR via AHL.

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

Result & Conclusion
GFP fluorescence increased in AHL-dose dependent manner, indicating that the hybrid promoter is enhanced by LuxR via AHL. Taken together with the graph shown in fig.1and formulation of Hill function fitting described above , the characteristics of the hybrid promoter expressed in Hill function, such as (n2,K2), is determined.

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Effect of LacI: IPTG assay

Fig. 2 Effect of LacI on hybrid promoter Enhanced fluorescence intensity represents down-regulation of LacI repression via IPTG.

Objective By testing how LacI represses lux-lac hybrid promoter, parameters(n3,K3) should be decided. In order to adjust effective concentration of LacI, IPTG was added.

Result & Conclusion GFP fluorescence intensity was enhanced in IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in Fig. 2 and the formulation of Hill function fitting described above, the characteristics of the hybrid promoter expressed in Hill function was determined.

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Appendix: Preliminary assays

We conducted preliminary assays as practice experiments and check of materials and methods.

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