Difference between revisions of "Team:Toulouse/Description/Eradicate"
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<ul> | <ul> | ||
<li><a href="#part1">Formate acaricide activity test</a></li> | <li><a href="#part1">Formate acaricide activity test</a></li> | ||
| − | <li><a href="#part2">How to synthesize formate with <i>E.coli</i>?</a></li> | + | <li><a href="#part2">How to synthesize formate with <i>E. coli</i>?</a></li> |
</ul> | </ul> | ||
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<p align="justify" style="font-size:15px;"> | <p align="justify" style="font-size:15px;"> | ||
| − | After being attracted, varroas will have to be eliminated. Beekeepers already use some treatments to fight against varroa, | + | After being attracted, varroas will have to be eliminated. Beekeepers already use some treatments to fight against varroa, like 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, | for bees and humans. Beside, varroa is developing resistance against some of these treatments, | ||
making them ineffective [1]. | making them ineffective [1]. | ||
<br> | <br> | ||
<br> | <br> | ||
| − | In the list of the molecules mentioned above, formate has already prove its acaricide property [2], and to our knowledge, no resistance has been reported yet. Formate also presents the advantage to be naturally synthesized by <i>E.coli</i>. | + | In the list of the molecules mentioned above, formate has already prove its acaricide property [2], and to our knowledge, no resistance has been reported yet. Formate also presents the advantage to be naturally synthesized by <i>E. coli</i> therefore is a natural molecule. |
<br> | <br> | ||
<br> | <br> | ||
Here, the project main goal will be to synthesize formate at the lowest concentration allowing an efficient elimination of varroa in the trap. The | Here, the project main goal will be to synthesize formate at the lowest concentration allowing an efficient elimination of varroa in the trap. The | ||
| − | bacteria will produce formate during a defined | + | bacteria will produce formate during a defined period, in order to reduce doses and minimize the toxicity on bees. |
</p> <div id="part1"></div> <!-- ANCHOR 1 --> <p align="justify" style="font-size:15px;"></p> | </p> <div id="part1"></div> <!-- ANCHOR 1 --> <p align="justify" style="font-size:15px;"></p> | ||
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In this experiment, three varroas were placed in a Petri dish containing | In this experiment, three varroas were placed in a Petri dish containing | ||
a cotton soaked with 400 µL of formate at different concentrations (50µM, 500µM, 1mM et 10mM). </p><div id="part2"></div> <!-- ANCHOR 2 --><p align="justify" style="font-size:15px;"> | a cotton soaked with 400 µL of formate at different concentrations (50µM, 500µM, 1mM et 10mM). </p><div id="part2"></div> <!-- ANCHOR 2 --><p align="justify" style="font-size:15px;"> | ||
| − | The experiment | + | The experiment ran for 6h and the varroas’ death was validated when no movement were detected even |
after a stimulus. The mite observation was performed with a binocular magnifier. | after a stimulus. The mite observation was performed with a binocular magnifier. | ||
<br> | <br> | ||
| Line 148: | Line 148: | ||
<div class="subtitle" > | <div class="subtitle" > | ||
| − | <h3>How to synthesize formate with <i>E.coli</i>?</h3> | + | <h3>How to synthesize formate with <i>E. coli</i>?</h3> |
</div> | </div> | ||
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<p align="justify" style="font-size:15px;"> | <p align="justify" style="font-size:15px;"> | ||
| − | Formate is a simple organic acid produced with | + | Formate is a simple organic acid that can be produced with <i>E. coli</i>. |
The initial substrate, glucose, is decomposed into pyruvate during | The initial substrate, glucose, is decomposed into pyruvate during | ||
| − | glycolysis, and formate is naturally synthesized | + | glycolysis, and formate is naturally synthesized by two key |
genes: | genes: | ||
</p> | </p> | ||
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<div class="group center"> | <div class="group center"> | ||
<p align="justify" style="font-size:15px;"> | <p align="justify" style="font-size:15px;"> | ||
| − | The | + | The key genes for formate synthesis were over-expressed. The genetic construction (<a href="https://static.igem.org/mediawiki/2015/b/bb/PUC57_-_Formate_synth.xdna.png"target="_blank">here</a>) was done by assembling the abovementioned genes, placed under the control of the P(Bla) constitutive promotor (BBa_I14018). RBS (BBa_B0030) were inserted to allow protein expression, and a strong terminator ended up the construction (BBa_B1006). We finally cloned these two genes and their RBS into a pSB1C3 vector (<a href="https://static.igem.org/mediawiki/2015/thumb/a/af/PSB1C3_Formate.xdna.png/715px-PSB1C3_Formate.xdna.png.jpeg"target="_blank">here</a>). |
</p> | </p> | ||
</div> | </div> | ||
Revision as of 16:49, 18 September 2015
Eradicate
Content
Treating Varroa destructor
After being attracted, varroas will have to be eliminated. Beekeepers already use some treatments to fight against varroa, like 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 has been reported yet. Formate also presents the advantage to be naturally synthesized by E. coli therefore is a natural molecule.
Here, the project main goal will be to synthesize formate at the lowest concentration allowing an efficient elimination of varroa in the trap. The
bacteria will produce formate during a defined 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 were placed in a Petri dish containing
a cotton soaked with 400 µL of formate at different concentrations (50µM, 500µM, 1mM et 10mM).
The experiment ran for 6h and the varroas’ death was validated when no movement were detected even
after a stimulus. The mite observation was performed with a binocular magnifier.
Figure 1: Formate miticide activity test
How to synthesize formate with E. coli?
Formate is a simple organic acid that can be produced with E. coli. The initial substrate, glucose, is decomposed into pyruvate during glycolysis, and formate is naturally synthesized by two key genes:
- pflB (Accession Number: EG10701) encoding the 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 (Accession Number: EG10028) encoding the 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 key genes for formate synthesis were over-expressed. The genetic construction (here) was done by assembling the abovementioned genes, placed under the control of the P(Bla) constitutive promotor (BBa_I14018). RBS (BBa_B0030) were inserted to allow protein expression, and a strong terminator ended up the construction (BBa_B1006). We finally cloned these two genes and their RBS into a pSB1C3 vector (here).
Formate pathway
READ MORE
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
