Team:UNITN-Trento/Project/Methyl Salicylate

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Results - Methyl Salicylate

It was difficult to find a volatile molecule that could be enzymatically produced by a bacterium and also demonstrated to be an efficient ripening inhbitor. There were not many candidates to choose from and after a long search we chose methyl salicylate (MeSA). Previous publications suggested that MeSA could inhibit the ripening of either kiwifruit (Aghdam M. et al., Journal of Agricultural Science. June 2011, Vol. 3, 2, pp. 149-156) and tomatoes, at a concentration of 0.5 mM ) (Ding, C. and Wang, Plant Science 2003, Y. 164 pp. 589-596.))

Fortunately many of the needed parts were already available because of the work of the MIT iGEM 2006 team (Eau de Coli).

Figure 1: In this picture is shown the pathway that was exploited to produce Methyl Salicyalte. The precursor is the chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like E.coli and B.subtilis ) have. The chorismate undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA and then the salicylate is obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism Pseudomonas aeruginosa . In the final part of the reaction, BMST1, a methyltransferase, transfer a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase.

We modified and improved these parts and resubmitted them to the registry, as they were not available in pSB1C3.

MeSA detection

To have a quantitative analysis we used a Finnigan Trace GC ULTRA with a flame ionization detector (FID) that allowed us to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM).

Then many measuraments were done with the instruments on different samples.

Figure 2: induced sample produces MeSA. A culture transformed with BBa_K1065102 was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to both samples. After 4 h the samples were connected to the Gas Chromatograph and a measure was taken. The chromatogram clearly shows how induced sample has a characteristic peak corresponding to methyl-salycilate.

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 			To have a quantitative analysis we used a Finnigan Trace GC ULTRA with a <b>flame ionization detector</b> (FID) that allowed us to detect ions formed during MeSA combustion in a hydrogen flame. The generation of this ions is proportional to MeSA concentration in the sample stream. A calibration curve was initially created using samples with a well known pure MeSA concentration (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM).
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+                      <img src="https://static.igem.org/mediawiki/2013/4/4f/Tn-2013_Taratura_MeSA.jpg">
+Then many measuraments were done with the instruments on different samples.
 		<img src="https://static.igem.org/mediawiki/2013/d/d4/Induced_sample_produce_MeSA.png">
 		<span class="tn-caption center" style="text-align:justify;"><b>Figure 2:</b> induced sample produces MeSA. A culture transformed with <a href="">BBa_K1065102</a> was grown until O.D. 0.6 was reached. The culture was then splitted in 2 samples and one was induced with 5 mM arabinose. 2 mM salycilic acid was added to both samples. After 4 h the samples were connected to the Gas Chromatograph and a measure was taken. The chromatogram clearly shows how induced sample has a characteristic peak corresponding to methyl-salycilate.</span>