Team:Toulouse/Description/Regulation
Regulate
Content
Regulate
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]
Light and Dark conditions
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 POmpC promoter .
In contrast, with light, PCB prevents the Cph8 autophosphorylation. OmpR will not be activated and the genes under POmpC 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 (BBa_K1587006) 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 PLac 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.
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References
- [1] Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM & Voigt CA (2005) Synthetic biology: Engineering Escherichia coli to see light. Nature 438: 441–442
- [2] Lee JM, Lee J, Kim T & Lee SK (2013) Switchable Gene Expression in Escherichia coli Using a Miniaturized Photobioreactor. PLoS ONE 8: e52382
- [3] Gambetta GA & Lagarias JC (2001) Genetic engineering of phytochrome biosynthesis in bacteria. PNAS 98: 10566–10571
