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

In order to respect the bee life cycle and to optimize our solution fighting Varroa destructor infestation of domestic bees, we have integrated a regulation system to our genetic construction: a day/night (or circadian) switch. In daytime, the bees are working outside to pollinate, and so, go back and forth at the beehive entrance, bringing potentially varroas inside the hive. In nighttime, bees are less active. Hence, we want our ApiColi to produce either butyric acid in daytime to attract varroa toward a 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 light.

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]

When there is no light, Cph8 autophosphorylates its EnvZ intracellular domain while consuming one molecule of ATP. The phosphoryl group will be subsequently transferred to the transcription factor OmpR, which then, will upregulate genes expressed from the OmpC promoter.
On the contrary, when there is light, PCB prevents the Cph8 autophosphorylation. Hence OmpR is not activated and there is no expression of genes under P_OmpC control.

This circadian switch is further enhanced with a combination of cI repressor and lacI repressor which enables ApiColi to produce butyric acid (polycistron B) when there is light, while formic acid is not produced, and conversely to produce formic acid (polycistron A) during the night and no butyric acid. Without light, polycistronic A genes, corresponding to the formate pathway, are transcribed The first protein, encoded by the cI gene downregulates the P_Lac promotor, hence, the polycistronic gene B is not transcribed. When there is light, there is no more expression of the polycistronic gene A, thus there is no more repression of the polycistronic gene B. Thus, there is production of butyrate via the expression of polycistronic gene B. In this same genetic element, there is a protein that represses the polycistronic gene A expression.