Team:Toulouse/Description/Regulation
Regulation
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. During the day, the bees are working outside doing pollination, so they make back and forth at the beehive entrance. This is when they can bring varroas into the hive. During the night they are less active. Hence, we want our ApiColi to produce butyric acid to attract varroa into the physical trap by day, and formic acid to kill it by night.
We based our reflexion on a light response system built in E. coli [1]
This system has then been used to design a genetic response
that can be switched on and off by light [2].
In our project, we have further enhanced the
process in order to be able to produce two
different genetic expressions 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 the result of the fusion between
the red light response domain of Cph1
(a phytochrome-like protein that comes
from Synechocystis sp PCC 6803) and the
intracellular histidin kinase EnvZ
(an osmolarity sensor protein) from E. coli.
Synthesis of PCB requires the expression of Ho1
(heme oxygenase gene) and PcyA (biliverdin reductase gene). [3]
Light and Dark conditions
When there is no light, Cph8 autophosphorylates its intracellular EnvZ domain while consuming one molecule of ATP. This activates the transcription factor OmpR by transferring its phosphate to it. OmpR-P therefore upregulates genes under control of the promoter POmpC.
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 PompC 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 pLac 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.
References
- [1] 2005 Nature, levskaya, synthetic biology: engineering E. coli to see light
- [2] 2013 PLOS, lee switchable gene expression in E. coli using a miniaturized photobioreactor
- [3] PNAS, 2001, gabetta, Genetic engineering of phytochrome biosynthesis in bacteria
