Difference between revisions of "Team:Toulouse/Description/Regulation"

In order to respect the bee life cycle and to optimize our solution to limit Varroa destructor infestation, we have integrated a regulation system to our genetic construction, in the form of a NOT logic gate controled by a day/night (or circadian) switch. In daytime, the bees go back and forth at the beehive entrance, bringing varroas inside or outside the hive. In nighttime, bees stay in the hive. Hence, we want our ApiColi E. coli strain to produce either butyric acid in daytime to attract varroa into the physical trap, or formic acid to kill it by night.

We based our reflexion on a light response system built in E. coli [1] The genetic system has been designed to be switched on and off in response to light [2]. In our project, we have further improved the process in order to control two alternative genetic programs depending on the light presence.

The core of the light sensor is composed of the membrane proteins PCB and Cph8. PCB is a chromophore (phycocyabilin) originating from cyanobacteria Synechocystis sp. PCC 6803. Cph8 is a hybrid protein between the red light response domain of Cph1 (a phytochrome-like protein from Synechocystis sp PCC 6803) and the intracellular domain of the histidin kinase EnvZ (an osmolarity sensor protein) from E. coli. The synthesis of PCB requires the expression of both Ho1 (heme oxygenase gene) and PcyA (biliverdin reductase gene). [3]

Whitout light, Cph8 autophosphorylates its EnvZ intracellular domain while consuming one molecule of ATP. The phosphoryl group will be subsequently transfered to the transcription factor OmpR, which then, will upregulate genes expressed from the P_OmpC promoter .

In contrast, with light, PCB prevents the Cph8 autophosphorylation. OmpR will not be activated and the genes under P_OmpC will not be expressed.

This circadian switch is further improved by integrating a NOT logic gate into the regulation circuitry. This is achieved using the phage lambda CI repressor as well as the bacterial LacI repressor. With such a design, in daytime, ApiColi will synthesize butyric acid (polycistron B) while repressing the synthesis of formic acid (polycistron A) and vice versa in nighttime. Without light, CI produced from the first gene of the polycistronic A, will repress the P_Lac promoter, preventing the expression of the polycistronic B genes whose first gene codes for the LacI repressor. In daytime, polycistronic A genes will not be expressed, there will be no CI repressor to prevent the transcription of the polycistronic B genes. Thus, butyrate will be produced and the LacI repressor will repress the polycistronic A genes.