Tokyo/Expression level check
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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.
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