Team:Groningen/project characterisation promoters prm
The modified lamdba Prm promoter (repressed by 434 cI) (BBa_I12006) plays a crucial role in our circuit. It is the promoter that induces the first memory unit. In order to be able to predict the behavior of these memory units, we have measured the activity of this promoter. As it is technically challenging to increase cI transcription factor concentration inside the cell directly, the promoter was induced indirectly. Promoter pBAD/AraC was induced with different concentrations of arabinose, which has resulted in production of the cI transcription factor that was cloned downstream from the promoter. Another construct, containing the modified promoter with GFP downstream of it was induced by the produced cI TF and the fluorescence was measured in a fluorimeter as described in protocols.
The modified lamdba Prm promoter (repressed by 434 cI) exhibits a dynamic response in a range of arabinose concentrations when induced indirectly. Both the speed of activation and its strength are higher with higher concentrations of the inducing molecule.
Because the promoter was not induced directly, the fluorescence intensity was compared to the predicted concentration of the cI transcription factor. The prediction was based on the measurements of pBAD/AraC promoter. We have assumed that the amount of GFP produced by the arabinose-induced promoter is directly related to that promoter activity and thus to concentration of any protein produced by this promoter when induced with different arabinose concentrations.
The effect of this comparison can be seen in Figure 3. The promoter activity grows exponentially with increasing cI concentrations. We believe that if we could achieve higher concentrations of cI, we would see a bend point and a second part of a sigmoid that would spread among a high concentration range.
Because of that this promoter is a good choice for constructing autoinducing loops and bistable switches.