Team:UNITN-Trento/Project/Fruit ripening

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<div class="container">
<div class="container">
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<div class="sheet">
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    <div class="sheet">
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<div>
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<h1>Fruit ripening</h1>
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<p>
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        <span class="tn-title">Fruit ripening</span>
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We have tested several type of fruit to verify that our engineered bacteria are able to produce <b>ethylene</b> (cell transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>) and <b>methyl salicylate</b> (cell transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>) for enhancing or blocking the ripening of fruit.
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        <p>
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</p>
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            We have tested several type of fruit to verify that our engineered bacteria are able to produce <b>ethylene</b> (<i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>) and <b>methyl salicylate</b> (cell transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>) for enhancing or slowing down the ripening of fruit, respectively.
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<p>
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        </p>
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The flasks containing the induced culture were kept at 37 degree in agitation and connected to a sealed jar (or a desiccator), where the fruit was exposed to either ethylene or methyl salicylate (MeSA). Negative controls were either a fruit kept in airtight jar or a fruit placed in a jar connected to a flask with non-induced cells.
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        <p>
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</p>
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            The flasks containing the induced culture were kept at 37 degree in agitation and connected to a sealed jar (or a desiccator) where the fruit was exposed to either ethylene or methyl salicylate (MeSA). Negative controls were either a fruit kept in a airtight jar or a fruit placed in a jar connected to a flask with non-induced <i>E. coli</i> NEB10&beta; cells.
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<p>
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        </p>
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Within the same experiment we used fruit assumed to be at the same ripening stage (i.e.: from the same bunch of bananas).
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        <p>
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</p>
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            Within the same experiment we used fruit assumed to be at the same ripening stage (i.e.: the same bunch of bananas).
 +
        </p>
 +
        <p>
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            The results obtained varied according to the fruit tested. We tested: bananas, plums, cherries and heirloom tomatoes, blackberries, kiwifruit and others. Results were evaluated <b>qualitatively</b> by observing <b>skin color changes</b> and <b>pulp texture or firmness</b>.
 +
        </p>
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        <img src="https://static.igem.org/mediawiki/2013/b/bd/Tn-2013_fruithood.jpg" />
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        <p>
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            In some cases we were able to detect different levels of fragrance, however this results were too subjective and we discarded them.
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        </p>
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        <p>
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            We were also able to perform a <b>iodine coloration test</b> on starch-containing fruit.
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        </p>
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        <p>
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            We also performed <b>quantitative</b> evaluations of ethylene effects by <b>fruit compression strength test</b> and by a <b>fruit color measurement test</b>.
 +
        </p>
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        <span class="tn-subtitle">Ethylene</span>
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        <p class="tn-msg">Click on the images to view them enlarged.</p>
 +
        <div id="jms-slideshow-1" class="jms-slideshow">
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<div id="ex1" class="step" data-scale="1" data-x="0" data-y="0" data-z="0">
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                <p align="center">
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                    Cherry tomatoes are successfully ripened
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                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
 +
                            <p>
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                                <span class="title">Type of fruit</span> Cherry tomatoes
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                            </p>
 +
                            <p>
 +
                                <span class="title">Duration of experiment</span> 8 days
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                            </p>
 +
                            <p>
 +
                                <span class="title">Exposition to Ethylene</span> 1 days
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                            </p>
 +
                            <p>
 +
                                <span class="title">Container</span> Fruit in 0.5 L jar
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                            </p>
 +
                        </td>
 +
                        <td>
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                            <a href=""><img src="https://static.igem.org/mediawiki/2013/7/7d/Tn-2013-fruit_pomodorini1.png"></a>
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                        </td>
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                    </tr>
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                </table>
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            </div>         
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</div>
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<div id="ex2" class="step" data-scale="1" data-x="0" data-y="0" data-z="5">
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                <p align="center">
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                    Ripened plums become red
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                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
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                            <p>
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                                <span class="title">Type of fruit</span> Plums
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                            </p>
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                            <p>
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                                <span class="title">Duration of experiment</span> 4 days
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                            </p>
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                            <p>
 +
                                <span class="title">Exposition to Ethylene</span> 4 days
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                            </p>
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                            <p>
 +
                                <span class="title">Container</span> Fruit in 1 L jar
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                            </p>
 +
                        </td>
 +
                        <td>
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                            <a href=""><img src="https://static.igem.org/mediawiki/2013/5/52/Tn-2013-fruit_susine1.png"></a>
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                        </td>
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                    </tr>
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                </table>
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            </div>
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 +
            <div id="ex3" class="step" data-scale="1" data-x="0" data-y="0" data-z="-10">
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                <p align="center">
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                    Heirloom tomatoes ready to be eaten
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                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
 +
                            <p>
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                                <span class="title">Type of fruit</span> Heirloom tomatoes
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                            </p>
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                            <p>
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                                <span class="title">Duration of experiment</span> 9 days
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                            </p>
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                            <p>
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                                <span class="title">Exposition to Ethylene</span> 9 days
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                            </p>
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                            <p>
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                                <span class="title">Container</span> Fruit in 1 L jar
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                            </p>
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                        </td>
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                        <td>
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                            <a href=""><img src="https://static.igem.org/mediawiki/2013/1/1b/Tn-2013-fruit_pomodorigrandi2.png"></a>
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                        </td>
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                    </tr>
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                </table>
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            </div>
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 +
            <div id="ex4" class="step" data-scale="1" data-x="0" data-y="0" data-z="-15">
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                <p align="center">
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                    Cherry tomatoes are successfully ripened
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                </p>
 +
                <table>
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                    <tr>
 +
                        <td>
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                            <p>
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                                <span class="title">Type of fruit</span> Cherry tomatoes
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                            </p>
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                            <p>
 +
                                <span class="title">Duration of experiment</span> 7 days
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                            </p>
 +
                            <p>
 +
                                <span class="title">Exposition to Ethylene</span> 1 day
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                            </p>
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                            <p>
 +
                                <span class="title">Container</span> Fruit in 0.5 L jar
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                            </p>
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                        </td>
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                        <td>
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                            <a href=""><img src="https://static.igem.org/mediawiki/2013/2/24/Tn-2013-fruit_pomodorini2.png"></a>
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                        </td>
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                    </tr>
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                </table>
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            </div>
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<div id="ex5" class="step" data-scale="1" data-x="0" data-y="0" data-z="-20">
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                <p align="center">
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                    Cavendish bananas are successfully ripened
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                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
 +
                            <p>
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                                <span class="title">Type of fruit</span> Cavendish banana
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                            </p>
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                            <p>
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                                <span class="title">Duration of experiment</span> 6 days
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                            </p>
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                            <p>
 +
                                <span class="title">Exposition to Ethylene</span> 6 days
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                            </p>
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                            <p>
 +
                                <span class="title">Container</span> Essicator (10.3L)
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                            </p>
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                        </td>
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                        <td>
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                            <a href=""><img src="https://static.igem.org/mediawiki/2013/3/39/Tn-2013-fruit-banana1.png"></a>
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                        </td>
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                    </tr>
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                </table>
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            </div>
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            <div id="ex6" class="step" data-scale="1" data-x="0" data-y="0" data-z="-25">
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                <p align="center">
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                    "Fried Green Tomatoes"
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                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
 +
                            <p>
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                                <span class="title">Type of fruit</span> Date tomatoes
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                            </p>
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                            <p>
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                                <span class="title">Duration of experiment</span> 7 days
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                            </p>
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                            <p>
 +
                                <span class="title">Exposition to Ethylene</span> 2 day w/, 3 days w/o, 2 days w/
 +
                            </p>
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                            <p>
 +
                                <span class="title">Container</span> Fruit in 0.5 L jar
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                            </p>
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                            <p>
 +
                                Note: Lycopene synthesis probably inhibited by the high temperature (&gt;30 degrees) in the lab as suggested in the literature
 +
                            </p>
 +
                        </td>
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                        <td>
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                            <a href=""><img src="https://static.igem.org/mediawiki/2013/5/5a/Tn-2013-fruit_datteri1.png"></a>
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                        </td>
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                    </tr>
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                </table>
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            </div>
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        </div>
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<div id="jms-slideshow" class="jms-slideshow">
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        <p>
 +
            In summary: <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> (our ethylene producing device) successfully ripen all the fruit tested when compared to the negative control, except for a few cases (Test with Date Tomatoes) where no significant effects were observed. We believe that this experiment did not work because the temperature in the laboratory was over 30 degrees, causing a decreased activity of lycopene synthesis enzymes <span class="tn-ref">(Mary Lu Arpaia et Al., Fruit Ripening &amp; Ethylene Management 2010, 9-10)</span>.
 +
        </p>
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    <div id="ex1" class="step" data-scale="1" data-x="0" data-y="0" data-z="0">
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        <span class="tn-sub-subtitle">Iodine Coloration Test</span>
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<table>
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        <p>
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<tr>
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            Starch accumulates in apples during growing season and is hydrolyzed to sugar in the last stages of maturation and development <span class="tn-ref">(Krotkov and Helson 1946)</span>. Hydrolysis occurs first in the core area and progresses outwards <span class="tn-ref">(Phillips and Poapst 1952)</span>. Starch in cut sections of the fruit reacts with a solution of iodine-potassium iodide to produce a blue-black color. The intensity and pattern of the color developed is an indication of the stage of maturation.<br>
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<td>
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        </p>
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<p>
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        <img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/07/Tn-2013-fruit-Apple_starch_test.png" />
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<span class="title">Type of fruit</span>
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        <span class="tn-caption"><b>Figure 1:</b> Starch test on apples. An apple was exposed to a culture transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> for three days and then left in a jar for other five days. It is well clear that treated apple is in a more advance ripening stage since iodine did not color the simple sugar produced from the hydrolisis of starch. Moreover we can assess a change in the pigment color (from green to red) of the treated apple.</span>
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Cavendish banana
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</p>
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<p>
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<span class="title">Duration of experiment</span>
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6 days
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</p>
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<p>
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<span class="title">Exposition to Ethylene</span>
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-
6 days
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</p>
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<p>
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<span class="title">Exposition to pure MeSA (LB-dissolved)</span>
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6 days
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</p>
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<p>
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<span class="title">Container</span>
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Essicator (10.3L)
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</p>
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<p>
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-
<span class="title">Comment</span>
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-
Maturation started 4 days after exposition to Ethylene. Methyl Salicylate in this experiment seems to ripen the fruit.  
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</p>
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</td>
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        <span class="tn-sub-subtitle">Fruit Compression Strength Test</span>
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<td>
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        <p>
-
<img src="https://static.igem.org/mediawiki/2013/3/39/Tn-2013-fruit-banana1.png" />
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            A compression test was used to determine the maturation stage of kiwifruit and bananas. All these measurements were done in the laboratory of eng. Alessandro Pegoretti in the Industrial Engineering Department. In this test the fruit is subjected to a crushing load to measure the resistence of a sample to compression strength. The test allows to measure the strength necessary to break the material analyzed, as an indication of maturation stage.
-
</td>
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        </p>
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</tr>
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        <div class="tn-doublephoto-wrap">
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</table>
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            <img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/e/ec/Tn-2013-fruit-Kiwi%27s_compressive_strength.png" />
-
    </div>
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            <img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/a/a5/Tn2013_photkiwi.JPG" />
 +
        </div>
 +
        <span class="tn-caption"><b>Figure 2:</b> Results of the compression test on kiwifruit in the left panel. After exposure to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K651001</a> for 8 days the fruit were cut into small cubes of the same size. For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene (in green) were significantly more ripen than the two negative controls used (no cells in blue, and non induced cells transofrmed with <a href="http://parts.igem.org/Part:BBa_K1065001">Bba_K1065001</a> in red), as demonstrated by the lower strength needed to compress the fruit. In the right panel the Instrom machinery used with a kiwi sample.</span>
-
    <div id="ex2" class="step" data-scale="1" data-x="0" data-y="0" data-z="-5">
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        <div class="tn-doublephoto-wrap">
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<table>
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            <img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/b/b0/Tn2013_tomphoto.JPG" />
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<tr>
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            <img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/9/9d/Tn-2013-fruit-Comparison_of_ultimate_strength_in_kiwi.png" />
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<td>
+
        </div>
 +
        <span class="tn-caption"><b>Figure 3:</b> In the left panel Thomas is waiting for our results. In the right panel, averages of ultimate strength for kiwifruit treated with ethylene and negative controls. Kiwifruit exposed to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and negative controls were left for 8 days in an airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimension from the same fruit. For a comparison we also estimated the strength based on the best 3 measurements obtained. A significant difference (25 fold less) between the treated and untreated kiwis is evident, indicating that our ethylene producing device (BBa_K1065001) successfully ripened the kiwifruit exposed to ethylene after 8 days.</span>
-
<p>
+
        <div class="tn-doublephoto-wrap">
-
<span class="title">Type of fruit</span>
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            <img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/8/80/Tn-2013-fruit-Banana%27s_compressive_strength.png" />
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Cherry plum
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            <img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/4/40/Tn-2013_brunophoto.JPG" />
-
</p>
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        </div>
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<p>
+
        <span class="tn-caption"><b>Figure 4:</b> In the left panel bananas compression test results. After exposure to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K651001</a> for 5 days the bananas were cut into small cubes of the same size. For each banana we tested 5 samples. Negative controls were treated and analysed with the same method. Banana exposed to ethylene (in green) were slightly more soft than the two negative controls used (no cells in blue, and non induced cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">Bba_K1065001</a> in red). In the right panel Bruno observes with curiosity the functioning of the machine.</span>
-
<span class="title">Duration of experiment</span>
+
-
4 days
+
-
</p>
+
-
<p>
+
-
<span class="title">Exposition to Ethylene</span>
+
-
4 days
+
-
</p>
+
-
<p>
+
-
<span class="title">Exposition to pure MeSA (LB-dissolved)</span>
+
-
4 days
+
-
</p>
+
-
<p>
+
-
<span class="title">Container</span>
+
-
Fruit in 1 L jar
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</p>
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-
<p>
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-
<span class="title">Comment</span>
+
-
Ethylene successfully ripened plums after XX days. Methyl salicylate in this experiment seems to ripen the fruit.
+
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</p>
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</td>
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        <div class="tn-doublephoto-wrap">
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<td>
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            <img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/6/65/Tn-2013_banana.JPG" />
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<img src="https://static.igem.org/mediawiki/2013/3/39/Tn-2013-fruit-banana1.png" />
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            <img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/8/80/Tn-2013-fruit-Comparison_of_ultimate_strength_in_banana.png" />
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</td>
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        </div>
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</tr>
+
        <span class="tn-caption"><b>Figure 5:</b> In the left panel, a banana's sample between the plate of machinery. In the right panel average of ultimate strength for bananas treated with ethylene and negative controls. Bananas exposed to ethylene produced by <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and negative controls were left for 5 days in a airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimensions from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained. Treated bananas are 1.5 fold less firm than untreated bananas.</span>
-
</table>
+
-
    </div>
+
-
</div>
+
        <p>
 +
            The test helped us to quantify the stage of maturation of kiwifruit and bananas, as another confirmation that our ethylene producing device works as predicted. However the test worked better on kiwis rather than bananas probably because the bananas used started already from a more advanced maturation stage.
 +
        </p>
-
<div>
+
        <span class="tn-sub-subtitle">Fruit color measurement test</span>
 +
        <p>
 +
            We used this test to quantify the difference between the color of ripe and unripe fruit's pulp. All measurments were done in the laboratory of eng. Sara Spilimbergo in the Industrial Engineering Department.
 +
        </p>
 +
        <p>
 +
            We estimated the color of kiwifruit using a high resolution miniature spectrometer, connected to a optical fiber.
 +
        </p>
 +
        <p>
 +
            The instrument measures three parameters L* (lightness), a* (redness) and b* (yellowness). The L* parameter indicate a range between black (0) and white (100), the a* parameter indicate a range between red (+a*) and green (-a*, complemental color of red), the b* parameter indicate a range between yellow (+b*) and blue (-b*, a complemental color of yellow). A shift in the color toward a darker green and a decrease in lightness are an indication of kiwi ripening.
 +
        </p>
 +
        <div class="tn-doublephoto-wrap">
 +
            <img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/f/fc/Tn-2013-fruit-Lightness_of_kiwifruit.png" />
 +
            <img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/a/ad/Tn-2013_kiwi1.jpg" />
 +
        </div>
 +
        <span class="tn-caption"><b>Figure 6:</b> In the left panel lightness measurament of kiwifruit treated for 8 days with ethylene (<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> induced, green) and negative controls (No cells, blue and <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> not induced, red). Treated kiwifuit has a lower L* value as a proof of advanced stage of ripening. In the right panel the instrument used to acquired the data. We note the high resolution miniature spectrometer with an optic fiber reflection probe connected.</span>
-
<p>
+
        <div class="tn-doublephoto-wrap">
-
The results obtained varied according to the fruit tested. We tested: bananas, cherry plums, cherries and heirloom tomatoes, plums, blackberries, kiwifruit, pears and others. Results were evaluated qualitatively by observing skin color changes and pulp texture or firmness. We were also able to perform a iodine coloration test on starch-containing fruit.
+
            <img class="photo no-bottom" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kiwi2.JPG" />
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</p>
+
            <img class="plot no-bottom" src="https://static.igem.org/mediawiki/2013/0/03/Tn-2013-fruit-Color_measurement_of_kiwifruit.png" />
-
<p>
+
        </div>
-
In some cases we were able to detect different levels of fragrance, however this results were too subjective and we discarded them. We also performed quantitative evaluations of ethylene effects by fruit compression strength test.
+
        <span class="tn-caption"><b>Figure 7:</b> In the left panel a detail of the fiber optic reflection probe and a slice-sample of kiwifruit. In the right panel the color measurement of kiwifruit. Average B* (yellow intensity) and average A* (red intensity) were calculated on 5 slices of the same kiwifruit. Kiwi treated with ethylene (<a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> induced, blue) showed a consistent shift toward a darker green shade respect to controls (no cells, green and <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_1065001</a> not induced, red).</span>
-
</p>
+
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<p>
+
-
In summary: cell transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> (our ethylene producing device) successfully ripen all the fruit tested when compared to the negative control, except for a few cases (Test with Date Tomatoes) where no significant effects were observed. We believe that in the first case the experiment did not work because the temperature in the laboratory (in those days) was over 30 degrees. We know the importance of temperature on ripening of vine-rape fruit (with external color). In our specific case the too high temperature (>25°C) caused inhibition of ethylene production which is needed for good lycopene synthesis. [reference]
+
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</p>
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<p>
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Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065102">BBa_K1065102</a> and <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> (our methyl salicylate producing devices) did not show any difference when compared to the negative control.  We were not able to determine if MeSA actually inhibited fruit ripening because the experiments were not long enough to allow ripening of the control. We demonstrated by XX that YY ml of bacteria culture (induced at O.D.600 equal to 0.6) transformed with our device produce successfully MeSA at a concentration of xx ppm in the culture medium. However, the final concentration of the inhibitor in the gas phase in the jar could be lower than the needed concentration. We are now in the process of better estimating these values.
+
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</p>
+
-
<p>
+
-
Exposure to pure MeSA instead caused an excessive ripening when added to the culture medium while it had a toxic effect when the fruit was drenched with it. MeSA was reported to slow down ripening at high concentration (XX) and to accelerate the process at lower concentration (YY). [ref] We think that under the used conditions its concentration was above the threshold required to inhibit ripening.
+
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</p>
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</div>
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<span class="tn-effect">We have engineered a device able to ripen different kind of fruit!</span>
 +
 
 +
        <span class="tn-subtitle">Methyl salicylate</span>
 +
 
 +
        <p class="tn-msg">Click on the images to view them enlarged.</p>
 +
        <div id="jms-slideshow-2" class="jms-slideshow">
 +
            <div id="ex12" class="step" data-scale="1" data-x="0" data-y="0" data-z="0">
 +
                <p align="center">
 +
                    Date tomatoes stay green
 +
                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
 +
                            <p>
 +
                                <span class="title">Type of fruit</span> Date tomatoes
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Duration of experiment</span> 7 days
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Exposition to MeSA <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>)</span> 2 day w/, 3 days w/o, 2 days w/
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Container</span> Fruit in 0.5 L jar
 +
                            </p>
 +
                        </td>
 +
                        <td>
 +
                            <a href=""><img src="https://static.igem.org/mediawiki/2013/3/3d/Tn-2013-fruit-datteri1_mesa.png"></a>
 +
                        </td>
 +
                    </tr>
 +
                </table>
 +
            </div>
 +
            <div id="ex22" class="step" data-scale="1" data-x="0" data-y="0" data-z="-5">
 +
                <p align="center">
 +
                    Cherry tomatoes unripen
 +
                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
 +
                            <p>
 +
                                <span class="title">Type of fruit</span> Cherry tomatoes
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Duration of experiment</span> 7 days
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Exposition to MeSA (<a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a>)</span> 7 days
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Container</span> Fruit in 0.5 L jar
 +
                            </p>
 +
                            <p>
 +
                                Note: One of the control was probably at a different maturation state ad day1.
 +
                            </p>
 +
                        </td>
 +
                        <td>
 +
                            <a href=""><img src="https://static.igem.org/mediawiki/2013/8/87/Tn-2013-fruit-pomodorini2_mesa.png"></a>
 +
                        </td>
 +
                    </tr>
 +
                </table>
 +
            </div>
 +
            <div id="ex32" class="step" data-scale="1" data-x="0" data-y="0" data-z="-10">
 +
                <p align="center">
 +
                    Pure MeSA has a toxic effect on cerry tomatoes
 +
                </p>
 +
                <table>
 +
                    <tr>
 +
                        <td>
 +
                            <p>
 +
                                <span class="title">Type of fruit</span> Cherry tomatoes
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Duration of experiment</span> 8 days
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Immersion in MeSA pure</span> 8 days
 +
                            </p>
 +
                            <p>
 +
                                <span class="title">Container</span> Fruit in 0.5 L jar
 +
                            </p>
 +
                            <p>
 +
                                Note: The immersion in MeSA pure killed the fruit.
 +
                            </p>
 +
                        </td>
 +
                        <td>
 +
                            <a href=""><img src="https://static.igem.org/mediawiki/2013/3/3c/Tn-2013-fruit-pomodorini1_mesa.png"></a>
 +
                        </td>
 +
                    </tr>
 +
                </table>
 +
            </div>
 +
        </div>
 +
 
 +
        <p>
 +
            <i>E. coli</i> NEB10&beta; cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065106">BBa_K1065106</a> (our methyl salicylate producing devices) did not show any difference when compared to the negative control. We were not able to determine if MeSA actually inhibited fruit ripening because the experiments were not long enough to allow ripening of the control. However, the final concentration of the inhibitor in the gas phase in the jar could be lower than the needed concentration. We are now in the process of better estimating these values.
 +
        </p>
 +
        <p>
 +
            Exposure to pure MeSA coused a toxic effect on cherry tomatoes. We also tested the effect of pure MeSA dissolved in LB but we obtain inconsistent results. For example in a few cases we observed an unexpected change color in the skin of plums and banana. MeSA was reported infact to slow down ripening at high concentration (0.5mM) and to accelerate the process at lower concentration (0.01mM) <span class="tn-ref">(Ding, C. and Wang, Y. 164,  Plant Science 2003, pp. 589-596)</span>. We think that under the used conditions its concentration was above the threshold required to inhibit ripening.<br>
 +
        </p>
 +
 
 +
       
 +
    </div>
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Latest revision as of 12:56, 15 October 2013

Fruit ripening

We have tested several type of fruit to verify that our engineered bacteria are able to produce ethylene (E. coli NEB10β cells transformed with BBa_K1065001) and methyl salicylate (cell transformed with BBa_K1065106) for enhancing or slowing down the ripening of fruit, respectively.

The flasks containing the induced culture were kept at 37 degree in agitation and connected to a sealed jar (or a desiccator) where the fruit was exposed to either ethylene or methyl salicylate (MeSA). Negative controls were either a fruit kept in a airtight jar or a fruit placed in a jar connected to a flask with non-induced E. coli NEB10β cells.

Within the same experiment we used fruit assumed to be at the same ripening stage (i.e.: the same bunch of bananas).

The results obtained varied according to the fruit tested. We tested: bananas, plums, cherries and heirloom tomatoes, blackberries, kiwifruit and others. Results were evaluated qualitatively by observing skin color changes and pulp texture or firmness.

In some cases we were able to detect different levels of fragrance, however this results were too subjective and we discarded them.

We were also able to perform a iodine coloration test on starch-containing fruit.

We also performed quantitative evaluations of ethylene effects by fruit compression strength test and by a fruit color measurement test.

Ethylene

Click on the images to view them enlarged.

Cherry tomatoes are successfully ripened

Type of fruit Cherry tomatoes

Duration of experiment 8 days

Exposition to Ethylene 1 days

Container Fruit in 0.5 L jar

Ripened plums become red

Type of fruit Plums

Duration of experiment 4 days

Exposition to Ethylene 4 days

Container Fruit in 1 L jar

Heirloom tomatoes ready to be eaten

Type of fruit Heirloom tomatoes

Duration of experiment 9 days

Exposition to Ethylene 9 days

Container Fruit in 1 L jar

Cherry tomatoes are successfully ripened

Type of fruit Cherry tomatoes

Duration of experiment 7 days

Exposition to Ethylene 1 day

Container Fruit in 0.5 L jar

Cavendish bananas are successfully ripened

Type of fruit Cavendish banana

Duration of experiment 6 days

Exposition to Ethylene 6 days

Container Essicator (10.3L)

"Fried Green Tomatoes"

Type of fruit Date tomatoes

Duration of experiment 7 days

Exposition to Ethylene 2 day w/, 3 days w/o, 2 days w/

Container Fruit in 0.5 L jar

Note: Lycopene synthesis probably inhibited by the high temperature (>30 degrees) in the lab as suggested in the literature

In summary: E. coli NEB10β cells transformed with BBa_K1065001 (our ethylene producing device) successfully ripen all the fruit tested when compared to the negative control, except for a few cases (Test with Date Tomatoes) where no significant effects were observed. We believe that this experiment did not work because the temperature in the laboratory was over 30 degrees, causing a decreased activity of lycopene synthesis enzymes (Mary Lu Arpaia et Al., Fruit Ripening & Ethylene Management 2010, 9-10).

Iodine Coloration Test

Starch accumulates in apples during growing season and is hydrolyzed to sugar in the last stages of maturation and development (Krotkov and Helson 1946). Hydrolysis occurs first in the core area and progresses outwards (Phillips and Poapst 1952). Starch in cut sections of the fruit reacts with a solution of iodine-potassium iodide to produce a blue-black color. The intensity and pattern of the color developed is an indication of the stage of maturation.

Figure 1: Starch test on apples. An apple was exposed to a culture transformed with BBa_K1065001 for three days and then left in a jar for other five days. It is well clear that treated apple is in a more advance ripening stage since iodine did not color the simple sugar produced from the hydrolisis of starch. Moreover we can assess a change in the pigment color (from green to red) of the treated apple. Fruit Compression Strength Test

A compression test was used to determine the maturation stage of kiwifruit and bananas. All these measurements were done in the laboratory of eng. Alessandro Pegoretti in the Industrial Engineering Department. In this test the fruit is subjected to a crushing load to measure the resistence of a sample to compression strength. The test allows to measure the strength necessary to break the material analyzed, as an indication of maturation stage.

Figure 2: Results of the compression test on kiwifruit in the left panel. After exposure to ethylene produced by BBa_K651001 for 8 days the fruit were cut into small cubes of the same size. For each fruit we tested 5 samples. Negative controls were treated and analysed with the same method. Kiwifruit exposed to ethylene (in green) were significantly more ripen than the two negative controls used (no cells in blue, and non induced cells transofrmed with Bba_K1065001 in red), as demonstrated by the lower strength needed to compress the fruit. In the right panel the Instrom machinery used with a kiwi sample.
Figure 3: In the left panel Thomas is waiting for our results. In the right panel, averages of ultimate strength for kiwifruit treated with ethylene and negative controls. Kiwifruit exposed to ethylene produced by BBa_K1065001 and negative controls were left for 8 days in an airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimension from the same fruit. For a comparison we also estimated the strength based on the best 3 measurements obtained. A significant difference (25 fold less) between the treated and untreated kiwis is evident, indicating that our ethylene producing device (BBa_K1065001) successfully ripened the kiwifruit exposed to ethylene after 8 days.
Figure 4: In the left panel bananas compression test results. After exposure to ethylene produced by BBa_K651001 for 5 days the bananas were cut into small cubes of the same size. For each banana we tested 5 samples. Negative controls were treated and analysed with the same method. Banana exposed to ethylene (in green) were slightly more soft than the two negative controls used (no cells in blue, and non induced cells transformed with Bba_K1065001 in red). In the right panel Bruno observes with curiosity the functioning of the machine.
Figure 5: In the left panel, a banana's sample between the plate of machinery. In the right panel average of ultimate strength for bananas treated with ethylene and negative controls. Bananas exposed to ethylene produced by BBa_K1065001 and negative controls were left for 5 days in a airtight jar and then subjected to a compression test. The reported values were calculated on a total of 5 samples of the same dimensions from the same fruit. For a comparison we also estimated the strength based the best 3 measurements obtained. Treated bananas are 1.5 fold less firm than untreated bananas.

The test helped us to quantify the stage of maturation of kiwifruit and bananas, as another confirmation that our ethylene producing device works as predicted. However the test worked better on kiwis rather than bananas probably because the bananas used started already from a more advanced maturation stage.

Fruit color measurement test

We used this test to quantify the difference between the color of ripe and unripe fruit's pulp. All measurments were done in the laboratory of eng. Sara Spilimbergo in the Industrial Engineering Department.

We estimated the color of kiwifruit using a high resolution miniature spectrometer, connected to a optical fiber.

The instrument measures three parameters L* (lightness), a* (redness) and b* (yellowness). The L* parameter indicate a range between black (0) and white (100), the a* parameter indicate a range between red (+a*) and green (-a*, complemental color of red), the b* parameter indicate a range between yellow (+b*) and blue (-b*, a complemental color of yellow). A shift in the color toward a darker green and a decrease in lightness are an indication of kiwi ripening.

Figure 6: In the left panel lightness measurament of kiwifruit treated for 8 days with ethylene (BBa_1065001 induced, green) and negative controls (No cells, blue and BBa_1065001 not induced, red). Treated kiwifuit has a lower L* value as a proof of advanced stage of ripening. In the right panel the instrument used to acquired the data. We note the high resolution miniature spectrometer with an optic fiber reflection probe connected.
Figure 7: In the left panel a detail of the fiber optic reflection probe and a slice-sample of kiwifruit. In the right panel the color measurement of kiwifruit. Average B* (yellow intensity) and average A* (red intensity) were calculated on 5 slices of the same kiwifruit. Kiwi treated with ethylene (BBa_1065001 induced, blue) showed a consistent shift toward a darker green shade respect to controls (no cells, green and BBa_1065001 not induced, red). We have engineered a device able to ripen different kind of fruit! Methyl salicylate

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Date tomatoes stay green

Type of fruit Date tomatoes

Duration of experiment 7 days

Exposition to MeSA BBa_K1065106) 2 day w/, 3 days w/o, 2 days w/

Container Fruit in 0.5 L jar

Cherry tomatoes unripen

Type of fruit Cherry tomatoes

Duration of experiment 7 days

Exposition to MeSA (BBa_K1065106) 7 days

Container Fruit in 0.5 L jar

Note: One of the control was probably at a different maturation state ad day1.

Pure MeSA has a toxic effect on cerry tomatoes

Type of fruit Cherry tomatoes

Duration of experiment 8 days

Immersion in MeSA pure 8 days

Container Fruit in 0.5 L jar

Note: The immersion in MeSA pure killed the fruit.

E. coli NEB10β cells transformed with BBa_K1065106 (our methyl salicylate producing devices) did not show any difference when compared to the negative control. We were not able to determine if MeSA actually inhibited fruit ripening because the experiments were not long enough to allow ripening of the control. However, the final concentration of the inhibitor in the gas phase in the jar could be lower than the needed concentration. We are now in the process of better estimating these values.

Exposure to pure MeSA coused a toxic effect on cherry tomatoes. We also tested the effect of pure MeSA dissolved in LB but we obtain inconsistent results. For example in a few cases we observed an unexpected change color in the skin of plums and banana. MeSA was reported infact to slow down ripening at high concentration (0.5mM) and to accelerate the process at lower concentration (0.01mM) (Ding, C. and Wang, Y. 164, Plant Science 2003, pp. 589-596). We think that under the used conditions its concentration was above the threshold required to inhibit ripening.

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