Team:Paris/Modeling/Protocol Of Characterization

Principles of the Experiments

To evaluate quantitativly the activity of a promoter in function of its transcription factors, we need datas in which the different values of the activities are correlated with various known and controlled values of the transcription factors concentrations. Therefore, we designed a generic plasmid in which the transcription factors are put under the control of previously characterized inducible promoter, and the studied promoter is put before a fluorescent reporter gene. In order to allow the study of the influence of two transcription factors over the tested pormoter, we chose to put the tested genes under two different inducible systems . One is the pBAD-AraC system. The second one is an indirect system were the gene is after the Tet inducible promoter « pTet ». The TetR gene would be expressed constitutively and at high rate thanks to a strong promoter (J23101) and its influence over the pTet promoter would be regulated by the concentration of the aTc molecule. That way the production of the tested gene can also be regulated. The J23101 and the pTet have been previously characterized. Here we show the design of two plasmids : one to test the influence of one gene and the other to test the influence of two genes over the tested promoter.

I-Plasmid for promoter characterization

I-A-For study with one Transcription Factor

I-B-For study with two Transcription Factor

The main design.

Assumptions Specific to the Experiments

The previous experiment is used to find parameters regarding to our modelization. However, the experiments themselves must be described in the same way to involve those parameters in a consistent way, and actually, to be interpreted.