Team:INSA-Lyon/Project/Stage3/Theory
From 2010.igem.org
Stage 3 : Regulation
The system of protein purification needs to be regulated to be fit. In fact, the cell machinery has limited energy sources to cope with the DNA replication, the proliferation, and simultaneously, the granule and the protein synthesis. We need to space out the synthesis of the different other elements.
As described in the article (Banki et al., Protein Science, 2005), we planned to induce first the granule synthesis during 30 hours and then to start the protein synthesis, which can bind directly to the granules already shaped. The rate of synthesis will be better if we stop the granules synthesis at this time. So we need promoters which turn each other ON and OFF.
We thought of two distinct systems in order to have an alternative if one of the promoters would not have been as good as expected.
First, we wanted to use the curli promoter in one of the system. We want to take advantage of the ability of this promoter to be ON at 28°C and OFF at higher temperatures. We looked for an iGEM promoter regulated by temperature, we chose a thermometer RNA. It is switched on above 32°C, allowing the transcription (BBa_K115017).
When the two constructs, functional, are in the same cell, the culture is moved at 28°C so that the PHB granules are formed under the control of Curli promoter. Then, the culture is moved at 37°C allowing the synthesis of the target proteins, activated by the thermometer RNA. At 37°C, the Curli promoter is off and the PHB granules are not synthesized anymore.
![FAS model](http://upload.wikimedia.org/wikipedia/en/6/64/FASmodel2.jpg)
FAS I model, extracted from Wikipedia, visualization by Kosi Gramatikoff.