Tokyo/Expression level check

From 2007.igem.org

Revision as of 04:18, 27 October 2007 by Kiga (Talk | contribs)


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