From 2007.igem.org

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

Parameters for the equations in Formulation <←link> have been experimentally determined in Assay1 <←link>. Analysing the result, the following experiments were turned out to be necessary.

Activation check by cell-produced AHL

Purpose

To check if worker E. coli (Sender) can produce enough AHL for our model to work by using different copy numbers of plasmids.

Result & Conclusion

Not only high copy number plasmid pSB1, also low copy number plasmid pSB4 and pBR produced enough AHL to activate the LacI hybrid promoter in other cells. Especially, pBR remarkably produced AHL in the present experiment.

⇒see more details

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

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.

⇒see more details