Team:Toulouse/Description/Eradicate
Eradicate
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Treating Varroa destructor
After being attracted, varroas will have to be eliminated. Beekeepers already use some treatments to fight against varroa, among them oxalate, fluvalinate, thymol or formate. All these treatments use high doses which are toxic for bees and humans. Beside, varroa is developing resistance against some of these treatments, making them ineffective [1]. In the list of the molecules mentioned above, formate has already prove its acaricide property [2], and to our knowledge, no resistance as been reported yet. Formate also present the advantage to be naturally synthesized by E.coli. For this part, the project main goal is to synthesize formate at low concentration. The bacteria will produce formate during a short time period, in order to reduce doses and minimize the toxicity on bees.
Formate acaricide activity test
Before formate production by our strain, it was necessary
to check the "miticide" activity of the molecule with a specific test.
In this experiment, three varroas are placed in a Petri dish containing
a cotton soaked with 400 µL of formate at different concentrations (50µM, 500µM, 1mM et 10mM).
The experiment runs for 6h and varroa’s death is validated when it does not move anymore even
after a stimulus. The mite observation is realized with a binocular magnifier.
Figure 1: Formate miticide activity test
How to synthesize formate with E.coli?
Formate is a simple organic acid produced with an E.coli strain. The initial substrate, glucose, is decomposed into pyruvate during glycolysis, and formate is naturally synthesized thanks to two key genes:
- pflB coding for pyruvate formate lyase which catalyzes the cleavage of pyruvate into C1 and C2. This enzyme is sensitive to oxygen and is only active in microaerobic or anaerobic conditions, which is the case within our device [3].
- pflA coding for pyruvate formate lyase activase, which is directly linked with the pyruvate formate lyase, enabling its activation [4].
Figure 3: Enzymatic reaction of the formate synthesis
The formate being naturally produced in E.coli, the key genes for synthesis are over-expressed. The genetic construction (here) is realized by assembling of these genes, which are placed under the control of P(Bla) constitutive promotor (BBa_I14018). Between genes there are ribosome binding sites (RBS)(BBa_B0030) to improve protein expression, and a strong terminator (BBa_B1006). We finally cloned this two genes and their RBS into a pSB1C3 vector (here).
Formate pathway
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References
- [1] Elzen PJ, Baxter JR, Spivak M & Wilson WT (2000) Control of Varroa jacobsoni Oud. resistant to fluvalinate and amitraz using coumaphos. Apidologie 31: 5
- [2]Satta A, Floris I, Eguaras M, Cabras P, Garau VL & Melis M (2005) Formic acid-based treatments for control of Varroa destructor in a Mediterranean area. J. Econ. Entomol. 98: 267–273
- [3] Becker A, Fritz-Wolf K, Kabsch W, Knappe J, Schultz S & Volker Wagner AF (1999) Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase. Nat. Struct. Biol. 6: 969–975
- [4] Crain AV & Broderick JB (2014) Pyruvate formate-lyase and its activation by pyruvate formate-lyase activating enzyme. J. Biol. Chem. 289: 5723–5729
