Team:UNITN-Trento/Project/Methyl Salicylate

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<h1>Results - Methyl Salicylate </h1>
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<span class="tn-title">Results - Methyl Salicylate </span>
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While the choice for the molecule to induce the ripening process was quiet easy because the quantities and the effects of ethylene on plants and on both climacteric and nonclimateric fruits are largely described in literature, was harder to find a synthesizable substance able to block the fruits’ ripening. This is due  to the fact that  most of the molecule reported to inhibit the maturation are toxic or they  have a terrible smell (this is the case of putrescine and cadaverin). Moreover we were searching for a volatile molecule in order to reach the fruit from the bacteria without contact of these two.
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Methyl salicylate it was our ripening inhibitor candidate. Differently from ethylene, the choice of the inhibitor was not easy, because it was difficult to find a volatile molecule, synthetizable enzymatically, that was also demonstrated to be an efficient ripening inhbitor.<br/><br/>
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After long discussions and researches we finally ran into some papers that present the effects  of Methyl Salicylate (MeSA) in inhibit fruit maturation. So, the choice to produce this organic ester was made.  
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After long discussions and researches we finally ran into some papers that present the effects  of Methyl Salicylate (MeSA) in inhibit fruit maturation (REF). In these studied MeSA was used succesfully as an inhibitor of Kiwifruit and tometoes ripening in a concentration of xx mM. We were excited to find out that in the registry were available many parts necessary for the biosynthesis of MeSA. The parts exploited are all from <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb">MIT iGEM team 2006 <i>Eau de Coli</i> project</a>.
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To produce it we have decided to exploit the work <i> Eau De Coli </i> done by the <a href="http://openwetware.org/wiki/IGEM:MIT/2006/Blurb"> MIT IGEM Team 2006 </a> that exploited the pathway shown in the picture that starts from chorismate, a metabolic intermediate of the Shikimate pathway. </p>
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  <img id="mesapath" style=" margin-bottom: 1em;" src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg" />
  <img id="mesapath" style=" margin-bottom: 1em;" src="https://static.igem.org/mediawiki/2013/d/dd/Tn-2013_MeSA_path.jpg" />
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We modified and improved these parts and resubmitted them to the registry, as they were not available in pSB1C3.
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In that project were designed BioBricks that contain all the three enzymes necessary for the production of MeSA: pCHA, the isochorismate synthase, pCHB, the isochorismate pyruvate lyase and BMST1, the SA methyltransferase- We have extracted them from the Registry Distribution Kit and we have exploited them to build our own devices as it is shown in the picture.
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<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg"/>
<img id="parts" src="https://static.igem.org/mediawiki/2013/4/41/Tn-2013_Mesa_main_parts.jpg"/>
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MeSA detection
MeSA detection
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At the beginning of our work we have worked with the device <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> that contained all the enzymes needed to obtain MeSA from the metabolic intermediate chorismate when arabinose was added. To detect the presence of the compound in the medium of our <i> E. coli </i> we tried both qualitative and quantitative analysis on sample.
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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.
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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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Sniff Test
 
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To immediately understand if the device worked we chose to exploit the typical wintergreen odor of Methyl Salicylate. To do this we performed three different SNIFF tests in which different samples were tested. In particular at each panelist were given two samples following a randomization scheme and it was asked if they could detect any differences between the two samples and to describe it. Here we have reported the result of one of the three where also samples of bacteria transformed with the device <a href="http://parts.igem.org/Part:BBa_K1065101">BBa_K1065101</a> were tested.
 
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As it is clearly visible the majority of people who have tried (almost 100 people) could recognized a difference and the presence of a’ balsamic’ odor in the samples where bacteria with the devices for MeSA production were present.
 
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GC-MS FID
 
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To have a quantitative analysis we used the Finnigan Trace GC ULTRA with a flame ionization detector (FID). To achieve the results shown here and to finally measure the quantity presents in our samples many tries were done. The column exploited was a DB5-MS capillary with a total length of 30 m (loops), an internal diameter of 0.25 mm and a film of 0.25 μm. The temperature program set for all the analyses was set to start with the oven thermostat and so the column at 80°C for 2’, to increase the temperature at the rate of 30°C/min until reaching the temperature of 280°C and maintaining it for 10’. The temperatures of the injector and of the FID were set 280°C. The carrier in the column was He 1.4 mL/min and the flow of the FID was set to be 40 mL/min of H2, 450 mL/min of air and to make up (it is needed for the stability of the helium flame) 30 mL/min of N2. The modality of the introduction of the samples in the detector was set to splitless, in particular 70 mL/min.  With these setting the retention index of MeSA was between 10.20-10.27 min.
 
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The measurements where performed on liquid and 1 μL of liquid from the samples was taken and injected in the instrument.  Here we have report the calibration curve that was constructed by using MeSA solutions with different concentrations (0 mM, 0.2 mM, 0.5 mM, 1.0 mM, 2 mM).
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After the calibration curve was constructed, the measurements on the MeSA producing bacteria were done: to not  damage the instrument, for the bacteria’s samples, the falcons were before centrifuged and 1 mL of supernatant was filtrated with a 0.22 μm filter and then 1 μL of each sample was injected in the gas chromatographer. The software that registered all the chromatograms was Finningan Xcalibur® after that the correct method was uploaded. This software also allowed to obtain directly the MeSA quantities from bacteria’s samples. In the histogram showed below you can see the results that we have obtained.
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Revision as of 09:32, 26 September 2013

Results - Methyl Salicylate

Methyl salicylate it was our ripening inhibitor candidate. Differently from ethylene, the choice of the inhibitor was not easy, because it was difficult to find a volatile molecule, synthetizable enzymatically, that was also demonstrated to be an efficient ripening inhbitor.

After long discussions and researches we finally ran into some papers that present the effects of Methyl Salicylate (MeSA) in inhibit fruit maturation (REF). In these studied MeSA was used succesfully as an inhibitor of Kiwifruit and tometoes ripening in a concentration of xx mM. We were excited to find out that in the registry were available many parts necessary for the biosynthesis of MeSA. The parts exploited are all from MIT iGEM team 2006 Eau de Coli project.

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).

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