Tokyo/Expression level check

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Revision as of 11:57, 25 October 2007

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

Activation check by cell-produced AHL 　　Expression level check on promoters + plasmid sets of A and B sides

Expression level check on two different promoters + plasmid sets

Purpose:

To measure and compare the activities of two different promoters + plasmid sets by fluorescence of GFP downstream of each promoter. Lambda cI-regulated promoter and the lux lac hybrid promoter were tested.

Samples:

・A4Δｐ　pc1-GFP
・A4 hybrid GFP PBR322TetR (+)AHL
・A4 hybrid GFP PBR322TetR (-)AHL
*The promoter pcI sequence was designed to contain OR1, -35, and -10 regions, but not R2 or OR3.

Procedure:

AHL assay Standard protocol
Wash
OD and fluorescence were measured 0, 3, and 6 hours after the fresh culture incubation started.

Result & Conclusion:

Two plasmid sets, A4Δｐ+pc1-GFP and A4 hybrid+GFP PBR322TetR (+)AHL, shows almost the same fluorescence of GFP, indicating that expression levels of both sets are almost the same though the latter is a bit smaller.
From the results the ranges of bistability and those of mono-stability are calculated as shown in Fig. 1.

Fig.1 Calculated coexistance stable region