Tokyo/Expression level check

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Revision as of 04:18, 27 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

Objective:

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:

Each sample cells has two plasmids.

• A4ΔP([http://partsregistry.org/Part:BBa_I751100 BBa_I751100]) / pc1-GFP ([http://partsregistry.org/Part:BBa_I751311 BBa_I751311])
• A4 hybrid promoter([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) / pBR322TetR (+)AHL
• A4 hybrid promoter([http://partsregistry.org/Part:BBa_I751101 BBa_I751101]) / 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 (Fig. 1).
In Fig2, this expression balance indicated by red line are compared to the ranges of bistability and those of mono-stability shown by the simulations. In the next construction, RBS and/ or promoter modifications are required to adjust the expression balance of the repressors.

Fig.1 Genes downstream of the promoters on the both sides were expressed to almost the same degrees in terms of GFP fluorescence.

Fig.2 Calculated coexistance stable region