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 data 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

II-Molecular design for Promoter Characterization Plasmid

Our aim is to make this plasmid useful not only for our project but for the whole iGEM comunity. This is why we decided to keep the Biobrick spirit as much as we could, making the plasmid compatible with the parts, so the teams using it need only the four traditional enzymes: EcoRI, XbaI, SpeI and PstI.We wanted also to make optional the introduction of a second tested gene. The strategy is then based in two plasmids. The principal plasmid contains everything needed to test the effect of one gene over the tested promoter activity. The second plasmid called « Accessory plasmid » can be introduced easily in the Principal Plasmid and contains the necessary elements to add the expression of a second gene to the system. The resulting plasmids are presented below.

II-A-Principal plasmid

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.