Team:UNITN-Trento/Project/Introduction

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Introduction
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        <span class="tn-title">Introduction</span>
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<span class="ita">
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        <span class="tn-quote">
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Have you ever thrown away some bananas because they were too ripe?
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            Have you ever thrown away some bananas because they were too ripe?<br>
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Are you one of many that hates waiting ages to eat the immature kiwis that you find at the supermarket?
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            Are you one of many that hates waiting ages to eat the immature kiwis that you find at the supermarket?<br>
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Do you know how banana and other fruits are picked green from the tree and arrive to the supermarket ready to be sold and eaten?
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            Do you know how bananas and other fruits are picked unripe from the tree and arrive to the supermarket ready to be sold and eaten?<br>
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Do you ever think of the huge fruit waste fruits in restaurants, markets, and industry?
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            Have you ever thought about how much fruit is wasted in restaurants, markets, and industry?
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</span>
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        </span>
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        <p>
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<p>We have decided to solve both these problems by designing and engineering a bacterial system able to control fruit's ripening in response to different stimulations: B. fruity.</p>
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            We have decided to solve both these problems by designing and engineering a bacterial system able to control fruit's ripening in response to different stimuli: <i>B. fruity</i>.
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        </p>
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<p>Furthermore, we have planned two different commercial products in order to eliminate waste of food and to make the consumption of these fruits accessible even in unusual places like schools and offices: the "<a href="">B. fruity Vending Machine</a>" and the "<a href="">B. fruity Home Edition</a>".</p>
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        <p>
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            Furthermore, we have planned two different commercial products in order to eliminate fruit waste and to make its consumption more accessible, even in unusual places like schools and offices: the "<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Vending%20Machine"><i>B. fruity </i>Vending Machine</a>" and the "<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Home%20Edition"><i>B. fruity </i>Home Edition</a>".
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<span class="subtitle">How does B. fruity work?</span>
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        </p>
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<p>We have designed an started to build a genetic circuit that activates fruit maturation thanks to ethylene production, a molecule produced by climateric fruits that affects growth, development, ripening, and senescence <span class="quote">(C. J. Brady, 1987)</span>. However, B. fruity does not exploit the complicated ethylene production pathway of plants, because of the undesirable production of hydrogen cyanide <span class="quote">(Shang Fa Yang et Al., 1984)</span>!!! We instead, decided to follow a different metabolic pathway that is present in <i>Pseudomonas syringae</i> which involves a single enzyme: 2-Oxoglutarate Oxygenase/Decarboxylase, commonly named the Ethylene Forming Enzyme (EFE).</p>
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        <span class="tn-subtitle">How does <i>B. fruity </i> work?</span>
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<p>To inhibit maturation we selected methyl salicylate, an ester also known as wintergreen oil, that is produced by many plants, as a defense mechanism, and was shown to slow down, if used at high concentration (5 mM), the ripening process in tomatoes <span class="quote">(Chang-Kui Ding et Al., 2002)</span>. To achieve methyl salicylate production we were lucky to use many of the parts submitted by the 2006 MIT iGEM team, as well as others built by us.</p>
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        <p>
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            We designed and started to build a genetic circuit that allows our bacteria to synthesize ethylene in order to boost fruit ripening. Ethylene is an hormone naturally produced by fruit and it affects growth, development, ripening, and senescence <span class="tn-ref">(C. J. Brady, Plant Physiology 1987, 38: 155-178)</span>. However, we did not engineer <i>B. fruity</i> to use the complicated ethylene synthesis pathway present in plants, because of the undesirable production of hydrogen cyanide <span class="tn-ref">(Shang Fa Yang et Al., Plant Physiol. 2001, 126(2): 742–749.). </span> Instead, we decided to follow a different metabolic pathway, present in <i>Pseudomonas syringae</i>, which involves only one enzyme: 2-Oxoglutarate Oxygenase/Decarboxylase, an Ethylene Forming Enzyme (EFE). For more information see the <a href =" https://2013.igem.org/Team:UNITN-Trento/Safety"> <b>Safety Page. </b> </a>
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<span class="subtitle">How is B. fruity activated?</span>
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        </p>
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<p>We have coupled this system to a blue light photoreceptor successfully used by other labs and iGEM teams in the past. Our system in the OFF state (no blue light) will produce methyl salicylate and stop unwanted ripening, while in the ON state ( Blue light exposure) it will produce ethylene and repress methyl salicylate production, thus promoting fruit ripening.</p>
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        <p>
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            As ripening inhibitor, we went for methyl salicylate (MeSA): an ester also known as wintergreen oil and naturally produced by many plants as a defense mechanism. It was previously shown to slow down the ripening process in tomatoes, at high concentration (5 mM) <span class="tn-ref">(Chang-Kui Ding et Al., Food Chemistry 2001, 76 213–218)</span>. To achieve its production we used parts submitted by the 2006 MIT iGEM team, as well as others which we built ourselves.
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<img style="display:block;width:100%;margin:auto;" src="https://static.igem.org/mediawiki/2013/5/5b/Tn-2013_intro_Efe_lineare.jpg">
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        </p>
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<span class="caption">Caption</span>
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        <span class="tn-quote-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info#q1">Do you know how plants produce ethylene?</a></span>
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<img style="display:block;width:100%;margin:auto;" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_intro_Mesa_lineare.jpg">
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        <span class="tn-quote-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info#q2">Do you know that ethylene is used commercially to ripen some fruits before they enter the market?</a></span>
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<span class="caption">Caption</span>
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        <span class="tn-quote-link"><a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info#q3">Do you know that fruit is generally classified in two main categories?</a></span>
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<p>You can check our <a href="">DATA page</a> for a full description of the circuit.</p>
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        <span class="tn-subtitle">How is <i>B. fruity </i> activated?</span>
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        <p>
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<span class="subtitle">Why B. fruity?</span>
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            We envisioned a system that is coupled to a blue light photoreceptor, the same receptor that has been used previously by other labs and iGEM teams. Our system in the OFF state (no blue light) will produce methyl salicylate and, in the absence of ethylene, will stop unwanted ripening (<b>Figure 2</b>), while in the ON state ( Blue light exposure) it will produce ethylene and repress methyl salicylate production, thus promoting fruit ripening (<b>Figure 1</b>).
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<p>We engineered the full system and characterized each component of the system in <i>Escherichia coli</i>. We have also tried to demonstrate the functionality of the enzymes involved in <i>Bacillus subtilis</i>.</p>
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        </p>
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        <p>
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<p>In order to develop a possible commercial product it is more desirable to use a chassis capable to resist for a certain amount of time without nutrients. So we thought that <i>Bacillus subtilis</i> could fit perfectly our purpose! It can make spores and it is easy to re-activate by removing the source of stress and adding, for example, water/nutrients. Moreover, <i>B. subtilis</i> is not a human pathogen. It can, however, degrade or may contaminate food, but rarely causes food poisoning. Therefore, with the right precautions and attention, this chassis appear to be the best system for our project.</p>
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            Thus far we have successfully built the device shown in <b>Figure 1</b> plus many other functional genetic constructs that you can find in the <a href="https://2013.igem.org/Team:UNITN-Trento/Parts">Parts page</a>.
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        </p>
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...Extras...
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        <img class="no-bottom" src="https://static.igem.org/mediawiki/2013/5/5b/Tn-2013_intro_Efe_lineare.jpg">
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        <span class="tn-caption"><b>Fig. 1:</b> a schematic representation of ethylene production regulated by a photo-inducible circuit. The inverter ensures that in presence of blue Light 2-Oxoglutarate Oxygenase/Decarboxylase (EFE) gene can be expressed.</span>
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        <img class="no-bottom" src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_intro_Mesa_lineare.jpg">
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        <span class="tn-caption"><b>Fig. 2:</b> a schematic representation of methyl salicylate production regulated by a photo-repressible circuit. Blue light blocks the blue receptor cassette inducing activity, resulting in the repression of MeSA production.</span>
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 +
        <p>
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            You can check our <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Datapage">DATA page</a> for a full description of the complete circuit.
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        </p>
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 +
        <span class="tn-subtitle">Why <i>B. fruity </i>?</span>
 +
        <p>
 +
            We engineered the full system and characterized each component of the system in <i>Escherichia coli</i>. We have also tried to demonstrate the functionality of the enzymes in <i>Bacillus subtilis</i>.
 +
        </p>
 +
        <p>
 +
            In order to develop a possible commercial product it is more desirable to use a chassis able to survive without nutrients for a longer time: we thought that <i>Bacillus subtilis</i> could fit perfectly our purpose! It can make spores and be easily re-activated by removing the source of stress and adding, for example, water/nutrients. Moreover, <i>B. subtilis</i> is not a human pathogen. Although this bacterium can degrade or contaminate food, with the right precautions this chassis seems to be the best system for our project.
 +
        </p>
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 +
        <span class="tn-effect">Follow our results to discover how we successfully ripen fruit!</span>
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        <img style="width:60%;"src="https://static.igem.org/mediawiki/2013/d/d5/Tn-2013_fruit_img_intro.JPG">
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        <br/>
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    </div>
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    <div class="sheet-2">
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        <a href="https://2013.igem.org/Team:UNITN-Trento">
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            <img class="tn-arr-prev" src="https://static.igem.org/mediawiki/2013/4/47/Tn-2013-arr-HOME_prev.png" />
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            <img src="https://static.igem.org/mediawiki/2013/6/6e/Tn-2013-arr-AAA_TOP.png" />
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        </a>
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        <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene">
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            <img class="tn-arr-next" src="https://static.igem.org/mediawiki/2013/d/d0/Tn-2013-arr-HOME_next.png" />
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<a id="tn-sp-tour" href="https://2013.igem.org/Team:UNITN-Trento/Project/Ethylene#tour">
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    <img src="https://static.igem.org/mediawiki/2013/6/6a/Tn-2013-tour-F_AAA_DSC_0054.png" />
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    <span>Continue the tour!</span>
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</a>
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Latest revision as of 12:42, 15 October 2013

Introduction Have you ever thrown away some bananas because they were too ripe?
Are you one of many that hates waiting ages to eat the immature kiwis that you find at the supermarket?
Do you know how bananas and other fruits are picked unripe from the tree and arrive to the supermarket ready to be sold and eaten?
Have you ever thought about how much fruit is wasted in restaurants, markets, and industry?

We have decided to solve both these problems by designing and engineering a bacterial system able to control fruit's ripening in response to different stimuli: B. fruity.

Furthermore, we have planned two different commercial products in order to eliminate fruit waste and to make its consumption more accessible, even in unusual places like schools and offices: the "B. fruity Vending Machine" and the "B. fruity Home Edition".

How does B. fruity work?

We designed and started to build a genetic circuit that allows our bacteria to synthesize ethylene in order to boost fruit ripening. Ethylene is an hormone naturally produced by fruit and it affects growth, development, ripening, and senescence (C. J. Brady, Plant Physiology 1987, 38: 155-178). However, we did not engineer B. fruity to use the complicated ethylene synthesis pathway present in plants, because of the undesirable production of hydrogen cyanide (Shang Fa Yang et Al., Plant Physiol. 2001, 126(2): 742–749.). Instead, we decided to follow a different metabolic pathway, present in Pseudomonas syringae, which involves only one enzyme: 2-Oxoglutarate Oxygenase/Decarboxylase, an Ethylene Forming Enzyme (EFE). For more information see the Safety Page.

As ripening inhibitor, we went for methyl salicylate (MeSA): an ester also known as wintergreen oil and naturally produced by many plants as a defense mechanism. It was previously shown to slow down the ripening process in tomatoes, at high concentration (5 mM) (Chang-Kui Ding et Al., Food Chemistry 2001, 76 213–218). To achieve its production we used parts submitted by the 2006 MIT iGEM team, as well as others which we built ourselves.

Do you know how plants produce ethylene? Do you know that ethylene is used commercially to ripen some fruits before they enter the market? Do you know that fruit is generally classified in two main categories? How is B. fruity activated?

We envisioned a system that is coupled to a blue light photoreceptor, the same receptor that has been used previously by other labs and iGEM teams. Our system in the OFF state (no blue light) will produce methyl salicylate and, in the absence of ethylene, will stop unwanted ripening (Figure 2), while in the ON state ( Blue light exposure) it will produce ethylene and repress methyl salicylate production, thus promoting fruit ripening (Figure 1).

Thus far we have successfully built the device shown in Figure 1 plus many other functional genetic constructs that you can find in the Parts page.

Fig. 1: a schematic representation of ethylene production regulated by a photo-inducible circuit. The inverter ensures that in presence of blue Light 2-Oxoglutarate Oxygenase/Decarboxylase (EFE) gene can be expressed. Fig. 2: a schematic representation of methyl salicylate production regulated by a photo-repressible circuit. Blue light blocks the blue receptor cassette inducing activity, resulting in the repression of MeSA production.

You can check our DATA page for a full description of the complete circuit.

Why B. fruity ?

We engineered the full system and characterized each component of the system in Escherichia coli. We have also tried to demonstrate the functionality of the enzymes in Bacillus subtilis.

In order to develop a possible commercial product it is more desirable to use a chassis able to survive without nutrients for a longer time: we thought that Bacillus subtilis could fit perfectly our purpose! It can make spores and be easily re-activated by removing the source of stress and adding, for example, water/nutrients. Moreover, B. subtilis is not a human pathogen. Although this bacterium can degrade or contaminate food, with the right precautions this chassis seems to be the best system for our project.

Follow our results to discover how we successfully ripen fruit!
Continue the tour!
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