Team:Toulouse/project/eradicate
Eradicate
Content
Treating varroa
After having attracted and trapped varroa in a confined device at the hive entrance, it is necessary to prevent it from escaping, and to eradicate it. It should be pointed out that beekeepers already use some treatments to fight against varroa, among them oxalate, fluvalinate, thymol or formate. All these known treatments use high doses which are toxic for bees and humans. On the other hand, varroa is developing resistance against these treatments, making them ineffective [1]. In the list of the molecules mentioned above, formate has already prove its acaricide property [2], and has the advantage to be naturally synthesized by E.coli. For this part, the project main goal is to synthesize formate at low concentration with the bacteria 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 cleavageog 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 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) to end this construction which is finally cloned into a pSB1C3 vector.
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: 437–441.
- [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. Journal of Economic Entomology 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.
