Difference between revisions of "Team:Toulouse/Description/Attract"

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Revision as of 09:28, 18 September 2015

iGEM Toulouse 2015

Attract


Content

How to attract Varroa destructor?

Just before capping, bee larvaes produce a wide range of molecules, those molecules warn the mite about the upcoming capping and motivate it to enter the cell [1]. Of all these molecules, scientific studies have shown that one can significantly attract varroa: butyrate [2].


Butyrate is a volatile acid which is non-toxic for honeybees nor for the human being, because it is already present at physiologic concentrations in their digestive tract. Moreover this molecule is naturally produced by some bacterial strains, which is an asset for identifying key enzymatic activities for this synthetic biology project [3].

Therefore, based on the patent US 8647615 (Figure 1), we decided to modify E. coli so it will synthesize butyrate in order to attract varroa [4].

Figure 1: Results of butyrate attraction test with quadrants method, US 8647615 B1 [4].

Butyrate attraction test

Figure 2: Butyrate attraction test using T tube, with varroa mite in the middle

To confirm this previous finding and make sure we can handle such experiment, a new protocol using a custom-made glass T-tube has been developed (Figure 2). In the first branch, there is a cotton soaked with 50 µL of water, in the second a cotton with 50 µL of butyrate at 4%, and finally the last one contains the varroa.

Butyrate being very volatile, our system used a pump to renew air, producing a concentration gradient as seen here.

How to produce butyrate with E.coli?

In this project, an Escherichia coli strain is used for its known simplicity of genetic manipulation and its adequacy with butyrate synthesis [5]. Indeed, among the five enzymes we selected to produce butyrate from acetyl-CoA, two enzymes are naturally produced by the bacteria. The following engineered butyrate pathway has been designed:


Figure 3: Engineered butyrate pathway


The initial substrate is glucose which is decomposed into acetyl-CoA during glycolysis. Butyrate pathway begins with acetyl-CoA: five genes are required with two homologous and three heterologous genes.


  • atoB present in E.coli (Accession Numbers: EG11672), coding for acetyl-CoA acetyltransferase, an acetyltransferase catalyzing the combination of two acetyl-CoA.


    Figure 4: Reaction catalyzed by acetyl-CoA acetyltransferase

  • hbd present in Clostridium acetobutylicum coding for 3-hydroxybutyryl-CoA dehydrogenase, an oxidoreductase catalyzing the formation of an alcohol function.


    Figure 5: Reaction catalyzed by 3-hydroxybutyryl-CoA dehydrogenase

  • crt present in C.acetobutylicum coding for 3-hydroxybutyryl-CoA dehydratase, a lyase cleaving carbon-oxygen bond.


    Figure 6: Reaction catalyzed by 3-hydroxybutyryl-CoA deshydratase

  • ccr present in Streptomyces collinus coding for crotonyl-CoA reductase, an oxidoreductase acting on CH=CH double bond. This enzyme is also in C.acetobutylicum with bcd gene coding for butyryl-CoA dehydrogenase, with the disadvantage to run with Electron Transfer Flavoprotein (ETF) which complicates the reaction [6].



    Figure 7: Reaction catalyzed by crotonyl-CoA reductase

  • tesB present in E.coli coding for acyl-CoA transferase 2, a thiolase which enables coenzyme A transfer.


    Figure 8: Reaction catalyzed by acyl-CoA transferase 2

Concerning heterologous genes (hbd, crt and ccr), a codon optimization has been performed in order to enable a good expression of these genes in E. coli. The genetic construction is then done by assembling the five genes presented earlier, which are placed under the control of P(Bla) constitutive promoter (BBa_I14018). In between the genes are placed ribosome binding sites (RBS) (BBa_B0030) to improve protein expression, and a strong terminator (BBa_B1006) is used to end this construction, which is to be cloned into a pSB1C3 vector (here).

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References


    [1] Le Conte Y, Arnold G, Trouiller J, Masson C, Chappe B & Ourisson G (1989) Attraction of the parasitic mite varroa to the drone larvae of honey bees by simple aliphatic esters. Science 245: 638–639
  • [2] Peter EA Teal, Adrian J. Duehl, Mark J. Carroll. The United States Of America, As Represented By The Secretary Of Agriculture. 2014. Methods for attracting honey bee parasitic mites, US 8647615 B1.
  • [3] Louis P & Flint HJ (2009) Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol. Lett. 294: 1–8
  • [4] Peter EA Teal, Adrian J. Duehl, Mark J. Carroll. The United States Of America, As Represented By The Secretary Of Agriculture. 2014. Methods for attracting honey bee parasitic mites, US 8647615 B1.
    • [5] Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T & Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering 10: 305–311
  • [6] Wallace KK, Bao ZY, Dai H, Digate R, Schuler G, Speedie MK & Reynolds KA (1995) Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli. Eur. J. Biochem. 233: 954–962