Team:UNITN-Trento/Project/Ethylene

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<div class="sheet">
<div class="sheet">
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<h1>
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<h1>Results - Ethylene</h1>
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Introduction
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<p>
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</h1>
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EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyzes ethylene synthesis from 2-Oxoglutarate, a TCA cycle intemediate molecule.
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<span class="quote">
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</p>
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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?<br/>
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<img style="box-shadow:none; margin-bottom:-1em;"src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" />
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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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<p>
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Have you ever thought about how much fruit is wasted in restaurants, markets, and industry?
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We characterized this gene in two chassis: <i>E. coli</i> and <i>B. subtilis</i>, using different contstructs that we designed.
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</p>
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<h2> EFE in <i>E. coli</i></h2>
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                <img src="https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts"/><br/>
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<p>
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In <i>E. coli</i>, EFE-catalyzed ethylene production was characterized using <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>, which is a composed part with EFE under the control of an AraC-pBAD promoter.
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</p>
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<h3>1. Ethylene detection</h3>
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<p>
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Ethylene production was detected using a Micro Gas Chromatograph (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#ethylene-detection-assay">protocol page</a> for the adopted methodology). The instrument was calibrated using two different air mixtures with well-defined quantities of each molecule (carbon dioxide, oxygen and ethylene).
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</p>
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<img src="https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" />
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<span class="caption">
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<b>Fig. 1:</b> Ethylene production. Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were grown in a thermoshaker until an O.D. of 0.5, placed in hermetically closed vial with a rubber septum and induced with 5 mM Arabinose. Ethylene was measured after 4 hours of induction at 37 &deg;C by connecting the vial to a Agilent Micro GC 3000.
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</span>
</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 stimuli: <i>B. fruity</i>.</p>
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To quantify the amount of ethylene produced the peak integral was converted into ppm.
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</p>
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<p>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="">B. fruity Vending Machine</a>" and the "<a href="">B. fruity Home Edition</a>".</p>
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<table id="ethylene_detected">
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<tr>
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<th class="center">Sample</th>
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<th class="center">Ethylene detected</th>
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</tr>
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<tr>
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<td>Not induced</td>
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<td class="right">0 &plusmn; 15 ppm</td>
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</tr>
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<tr>
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<td>Induced V = 1.5 ml</td>
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<td class="right">61 &plusmn; 15 ppm</td>
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</tr>
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<tr>
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<td>Induced V = 3 ml</td>
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<td class="right">101 &plusmn; 15 ppm</td>
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</tr>
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</table>
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<span class="caption center">
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<b>Table. 1:</b> ethylene detected quantities.
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</span>
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<span class="subtitle">How does B. fruity work?</span>
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<h3>2. Kinetic assay for ethylene production</h3>  
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<p>We designed and started to build a genetic circuit that allows our bacteria to synthesize ethylene in order to boost fruit maturation. Ethylene is a molecule naturally produced by fruit and it affects growth, development, ripening, and senescence. <span class="ref">(C. J. Brady, 1987)</span>
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<p>
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We performed a kinetic assay in order to analyze ethylene production over time (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for the adopted method).
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</p>
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<div class="plot_photo_wrapper">
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<img class="plot" style="width:66%!important;" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" />
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<img class="photo" style="width:33%!important;" src="https://static.igem.org/mediawiki/2013/9/98/Tn-2013_ethylene_kinetic_img.JPG" />
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</div>
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<span class="caption">
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<b>Fig. 2:</b> ethylene production (ppm) over time (min) of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and induced with 5 mM Arabinose  at different O.D.600 and cultured in different conditions. The control (not-induced sample) did not show any amount of ethylene.
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</span>
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However, <i>B. fruity</i> does not exploit the complicated pathway present in plants, because of the undesirable production of hydrogen cyanide!!! <span class="ref">(Shang Fa Yang et Al., 1984)</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).</p>
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<p>
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Figure 2 shows that induction of the culture at O.D.600 equal to 0.8 caused a 2-fold increase in ethylene production.
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</p>  
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<h3>3. Toxicity test</h3>
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<p>
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A toxicity test was performed inducing EFE expression with 5 mM arabinose. The growth curve was then compared to a non-induced sample.
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</p>
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<img src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" />
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<span class="caption center">
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<b>Fig. 3:</b> growth curves of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls.
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</span>
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<p>
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As expected, induced samples showed a decreased growth rate.
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</p>
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<div class="separator"></div>
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<h2>
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EFE under the control of a Blue light circuit in <i>E. coli</i>
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</h2>
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<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts"/>
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<p>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="ref">(Chang-Kui Ding et Al., 2002)</span> To achieve its production we used parts submitted by the 2006 MIT iGEM team, as well as others which we built ourselves.</p>
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<p>
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<span class="quote">
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To build our final system we placed EFE under the control of a photoinducible circuit.  We assembled the photoinducible circuit exploiting many subparts from different teams (Uppsala2011 and Berkeley 2006). The construct <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> includes an inverter that allows ethylene production only in presence of light. For more details on the design anc characterization of the circuit check the Blue light page of our wiki.
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<a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info">Do you know how plants produce ethylene?</a>
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</p>
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<h3>
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Photoinduced ethylene production - kinetic assay
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</h3>
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 +
<p>
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We performed a kinetic assay in order to analyze ethylene production over time using (<a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>). When the culture reached an OD of 0.7, it was placed in a hermetically closed vial and exposed to a blue light led (470 nm) while it was connected to the micro GC (see the protocol page for the adopted method).
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</p>
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<div class="plot_photo_wrapper">
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<img class="plot" src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" />
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<img class="photo"  src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" />
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</div>
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<span class="caption">
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<b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue led light over time (min) of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>.
</span>
</span>
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<span class="quote">
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<a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info">Do you know that ethylene is used commercially to ripen some fruits before they enter the market?</a>
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<h2>EFE in <i>B. subtilis</i></h2>
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</span>
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<img src="https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/>
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<span class="quote">
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<a href="https://2013.igem.org/Team:UNITN-Trento/Fruit_Info">Do you know that fruit is generally classified in two main categories?</a>
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</span>
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<span class="subtitle">How is B. fruity activated?</span>
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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, in the absence of ethylene, 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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<img src="https://static.igem.org/mediawiki/2013/5/5b/Tn-2013_intro_Efe_lineare.jpg">
 
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<span class="caption"><strong>Fig. 1:</strong> 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 src="https://static.igem.org/mediawiki/2013/7/79/Tn-2013_intro_Mesa_lineare.jpg">
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<h1>
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<span class="caption"><strong>Fig. 2:</strong> 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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Ethylene experiment - Summary
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</h1>
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<p>You can check our <a href="https://2013.igem.org/Team:UNITN-Trento/Project/Datapage">DATA page</a> for a full description of the circuit.</p>
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<p>
-
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2-Oxoglutarate Oxygenase/Decarboxylase (EFE), is a very powerfull enzyme that has been successfully characterized. At the end of a set of experiments we achieved the following results:
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<span class="subtitle">Why B. fruity?</span>
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</p>
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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 in <i>Bacillus subtilis</i>.</p>
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<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 but can, however, degrade or may contaminate food and only rarely cause food poisoning. Therefore, with the right precautions, this chassis seems to be the best system for our project.</p>
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<ul>
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<span style="text-align:center;" class="quote">
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<li>
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<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit_ripening">Check our fruit tests results!</a>
+
EFE was expressed under the control of an Arabinose inducible promoter in <i>E. coli</i>;
 +
</li>
 +
<li>
 +
ethylene was detected at the micro gas chromatograph and a quantitative kinetic curve was registered;
 +
</li>
 +
<li>
 +
EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production; 
 +
</li>
 +
<li>
 +
EFE was expressed in <i>B. subtilis</i> under the control of two different inducible promoters. Ethylene was not detected but induced samples showed a particular smell. Further analysis demostrated that induced samples reacted with lead-acetate paper strips, indicating the presence of sulfur compounds. Probably <i>B. subtilis</i> is capable of converting rapidly ethylene in other compounds; 
 +
</li>
 +
<li>
 +
our system was succcessfully exploited to accelerate fruit ripening.
 +
</li>
 +
</ul>
 +
<br/>
 +
<span class="quote">
 +
<a href="https://2013.igem.org/Team:UNITN-Trento/Project/Fruit_ripening">Check how we exploited <i>B. fruity</i> to ripen fruit!</a>
</span>
</span>
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</div>
</div>
</div>
</div>
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Revision as of 10:36, 19 September 2013

Results - Ethylene

EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyzes ethylene synthesis from 2-Oxoglutarate, a TCA cycle intemediate molecule.

Ethylene pathway

We characterized this gene in two chassis: E. coli and B. subtilis, using different contstructs that we designed.

EFE in E. coli

E. coli EFE parts

In E. coli, EFE-catalyzed ethylene production was characterized using BBa_K1065001, which is a composed part with EFE under the control of an AraC-pBAD promoter.

1. Ethylene detection

Ethylene production was detected using a Micro Gas Chromatograph (see the protocol page for the adopted methodology). The instrument was calibrated using two different air mixtures with well-defined quantities of each molecule (carbon dioxide, oxygen and ethylene).

Ethylene chromatogram Fig. 1: Ethylene production. Cells transformed with BBa_K1065001 were grown in a thermoshaker until an O.D. of 0.5, placed in hermetically closed vial with a rubber septum and induced with 5 mM Arabinose. Ethylene was measured after 4 hours of induction at 37 °C by connecting the vial to a Agilent Micro GC 3000.

To quantify the amount of ethylene produced the peak integral was converted into ppm.

Sample Ethylene detected
Not induced 0 ± 15 ppm
Induced V = 1.5 ml 61 ± 15 ppm
Induced V = 3 ml 101 ± 15 ppm
Table. 1: ethylene detected quantities.

2. Kinetic assay for ethylene production

We performed a kinetic assay in order to analyze ethylene production over time (see the protocol page for the adopted method).

kinetic_EFE_plot
Fig. 2: ethylene production (ppm) over time (min) of cells transformed with BBa_K1065001 and induced with 5 mM Arabinose at different O.D.600 and cultured in different conditions. The control (not-induced sample) did not show any amount of ethylene.

Figure 2 shows that induction of the culture at O.D.600 equal to 0.8 caused a 2-fold increase in ethylene production.

3. Toxicity test

A toxicity test was performed inducing EFE expression with 5 mM arabinose. The growth curve was then compared to a non-induced sample.

Toxicity test plot Fig. 3: growth curves of cells transformed with BBa_K1065001 and of controls.

As expected, induced samples showed a decreased growth rate.

EFE under the control of a Blue light circuit in E. coli

e.coli_bluelight-EFE_parts

To build our final system we placed EFE under the control of a photoinducible circuit. We assembled the photoinducible circuit exploiting many subparts from different teams (Uppsala2011 and Berkeley 2006). The construct BBa_K1065311 includes an inverter that allows ethylene production only in presence of light. For more details on the design anc characterization of the circuit check the Blue light page of our wiki.

Photoinduced ethylene production - kinetic assay

We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065311). When the culture reached an OD of 0.7, it was placed in a hermetically closed vial and exposed to a blue light led (470 nm) while it was connected to the micro GC (see the protocol page for the adopted method).

EFE-blue_light_plot
Fig. 4: Ethylene production (ppm) upon photoinduction with a blue led light over time (min) of cells transformed with BBa_K1065311.

EFE in B. subtilis

Ethylene experiment - Summary

2-Oxoglutarate Oxygenase/Decarboxylase (EFE), is a very powerfull enzyme that has been successfully characterized. At the end of a set of experiments we achieved the following results:

  • EFE was expressed under the control of an Arabinose inducible promoter in E. coli;
  • ethylene was detected at the micro gas chromatograph and a quantitative kinetic curve was registered;
  • EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production;
  • EFE was expressed in B. subtilis under the control of two different inducible promoters. Ethylene was not detected but induced samples showed a particular smell. Further analysis demostrated that induced samples reacted with lead-acetate paper strips, indicating the presence of sulfur compounds. Probably B. subtilis is capable of converting rapidly ethylene in other compounds;
  • our system was succcessfully exploited to accelerate fruit ripening.

Check how we exploited B. fruity to ripen fruit!
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