First of all, we needed a system that could cause a change in its medium conductivity. An extrusion pump seemed to be the best way to achieve this. Once this was devised, we needed a way to regulate the system. We decided to use a negative regulator because it's the only way to transcriptionally regulate the expression of a gene in a definitive way.
We had to be able to restart our system, so we could add a signal at anytime. This could be accomplished with an induction signal that disappeared rapidly after its involvement. The need of a link between the inductor signal and the repressor, lead us to include a little regulation cascade. This cascade allows us to add new steps which might increase our system’s complexity.
The components selected to fulfill the system requirements are enlisted in the next table:
Device BBa_K119009: The extrusion pump.
Our aim is to manipulate the transcription of rcnA by an inhibitory signal while maintaining the natural regulation of rcnA through RcnR. To achieve this the device contains a CI dependent promoter, RcnR binding site and the RcnA extrusion pump inserted in the vector pBBR1MCS-5.
Devices BBa_K119010/BBa_K119011: The regulatory device
In order to control the RcnA activity this device includes the gene encoding LuxR under the regulation TetR constitutive promoter followed by cI, which will repress RcnA in the prescence of AHL:LuxR. The last component of the device is the gene encoding AiiA. In BBa_K119010 lacZ promoter is upstream of AiiA, while BBa_K119011 carries a mutated version of it. The plasmid carrying this device will be PRK415.
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