Team:UNITN-Trento/Project/Ethylene

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<h1>Results - Ethylene</h1>
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<h1>Results - Ethylene</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. 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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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. 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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    <img src="https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" />
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<h2>E.coli</h2>
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<h2>E.coli</h2>
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In <i>E. coli</i>, EFE-catalyzed ethylene production was characterized using BBa_K1065001, which is a composed part with EFE under the AraC-pBAD promoter.
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In <i>E. coli</i>, EFE-catalyzed ethylene production was characterized using BBa_K1065001, which is a composed part with EFE under the AraC-pBAD promoter.
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<h3>1. Toxicity test</h3>
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<h3>1. Toxicity test</h3>
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A toxicity test was performed inducing EFE expression with 5 mM arabinose. The growth curve was then compared to a not-induced sample.
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A toxicity test was performed inducing EFE expression with 5 mM arabinose. The growth curve was then compared to a not-induced sample.
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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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<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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<b>Fig. 1:</b> growth curves of cells transformed with BBa_K1065001 and of controls.
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<b>Fig. 1:</b> growth curves of cells transformed with BBa_K1065001 and of controls.
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As expected, induced samples showed a decreased growth rate.
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As expected, induced samples showed a decreased growth rate.
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<h3>2. Ethylene detection</h3>
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<h3>2. Ethylene detection</h3>
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Then, ethylene 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.
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Then, ethylene 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.
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<img src="" alt="Ethylene chromatogram" />  
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<img src="" alt="Ethylene chromatogram" />  
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<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A.
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<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A.
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The chromatogram clearly shows the presence of a peak corresponding to ethylene; the peak integral was converted to ppm.
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The chromatogram clearly shows the presence of a peak corresponding to ethylene; the peak integral was converted to ppm.
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<table id="ethylene_detected">
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<table id="ethylene_detected">
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<th class="center">Sample</th>
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<th class="center">Sample</th>
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<th class="center">Ethylene detected</th>
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<th class="center">Ethylene detected</th>
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<td>Not induced</td>
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<td>Not induced</td>
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<td class="right">0 &plusmn; 15 ppm</td>
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<td class="right">0 &plusmn; 15 ppm</td>
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<td>Induced V = 1.5 ml</td>
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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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<td class="right">61 &plusmn; 15 ppm</td>
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<td>Induced V = 3 ml</td>
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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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<td class="right">101 &plusmn; 15 ppm</td>
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<span class="caption center">
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<b>Table. 1:</b> ethylene detected quantities.
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<b>Table. 1:</b> ethylene detected quantities.
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<h3>3. Kinetic assay for ethylene production</h3>  
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<h3>3. Kinetic assay for ethylene production</h3>  
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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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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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<img 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 src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" />
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<span class="caption">
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<b>Fig. 3:</b> ethylene production (ppm) over time (min) of cells induced at different O.D.600 and cultured at different environmental conditions.
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<b>Fig. 3:</b> ethylene production (ppm) over time (min) of cells induced at different O.D.600 and cultured at different environmental conditions.
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Figure 3 shows how inducing the culture at O.D.600 equal to 0.8 a.u. caused a 2-fold increase in ethylene production.
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Figure 3 shows how inducing the culture at O.D.600 equal to 0.8 a.u. caused a 2-fold increase in ethylene production.
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<div class="separator"></div>
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<span class="quote">
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Do you know how plants produce ethylene?
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Do you know how plants produce ethylene?
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Plants naturally produce ethylene starting from a common amino acid: methionine (MET), which is transformed in S-adenosyl-methionine (SAM) by a reaction catalyzed by SAM synthetase. The key enzyme of the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid (ACC), the immediate precursor of ethylene. Quite recently, the final enzyme of the pathway was identified: ACC oxidase, which converts ACC to ethylene.
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Plants naturally produce ethylene starting from a common amino acid: methionine (MET), which is transformed in S-adenosyl-methionine (SAM) by a reaction catalyzed by SAM synthetase. The key enzyme of the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid (ACC), the immediate precursor of ethylene. Quite recently, the final enzyme of the pathway was identified: ACC oxidase, which converts ACC to ethylene.
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<img src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg" alt="plants_eth_path" />
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<img src="https://static.igem.org/mediawiki/2013/b/bb/Tn-2013-project_ethylene-Plants_path.jpg" alt="plants_eth_path" />
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<span class="caption">
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<strong>Fig. 4:</strong> schematic view of ethylene producing pathway I (plants).
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<strong>Fig. 4:</strong> schematic view of ethylene producing pathway I (plants).
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<span class="quote">
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Do you know that ethylene is used commercially to ripen some fruits before they enter the market?
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Do you know that ethylene is used commercially to ripen some fruits before they enter the market?
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All fruits, with a few exceptions (European pears, avocados and bananas), reach their best eating quality when allowed to ripen on the tree or plant. However, some fruits are usually picked mature but unripe so that they can withstand the post-harvest handling system when shipped long-distance. Bananas, for example, are treated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Treatment of avocados, kiwi fruit, mangos and pears before marketing is increasingly being performed to provide consumers with the choiche of purchasing ready-to-eat, ripe fruits or mature fruits that can be ripened at home. This practice has, in many cases, resulted in increased sales and profits.
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All fruits, with a few exceptions (European pears, avocados and bananas), reach their best eating quality when allowed to ripen on the tree or plant. However, some fruits are usually picked mature but unripe so that they can withstand the post-harvest handling system when shipped long-distance. Bananas, for example, are treated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Treatment of avocados, kiwi fruit, mangos and pears before marketing is increasingly being performed to provide consumers with the choiche of purchasing ready-to-eat, ripe fruits or mature fruits that can be ripened at home. This practice has, in many cases, resulted in increased sales and profits.
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Revision as of 20:23, 11 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. We characterized this gene in two chassis: E. coli and B. subtilis, using different contstructs that we designed.

E.coli

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

1. Toxicity test

A toxicity test was performed inducing EFE expression with 5 mM arabinose. The growth curve was then compared to a not-induced sample.

Toxicity test plot Fig. 1: growth curves of cells transformed with BBa_K1065001 and of controls.

As expected, induced samples showed a decreased growth rate.

2. Ethylene detection

Then, ethylene 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.

Ethylene chromatogram Fig. 2: chromatogram obtained using an Agilent Micro GC 3000A.

The chromatogram clearly shows the presence of a peak corresponding to ethylene; the peak integral was converted to 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.

3. 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. 3: ethylene production (ppm) over time (min) of cells induced at different O.D.600 and cultured at different environmental conditions.

Figure 3 shows how inducing the culture at O.D.600 equal to 0.8 a.u. caused a 2-fold increase in ethylene production.

Do you know how plants produce ethylene?

Plants naturally produce ethylene starting from a common amino acid: methionine (MET), which is transformed in S-adenosyl-methionine (SAM) by a reaction catalyzed by SAM synthetase. The key enzyme of the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid (ACC), the immediate precursor of ethylene. Quite recently, the final enzyme of the pathway was identified: ACC oxidase, which converts ACC to ethylene.

plants_eth_path Fig. 4: schematic view of ethylene producing pathway I (plants). Do you know that ethylene is used commercially to ripen some fruits before they enter the market?

All fruits, with a few exceptions (European pears, avocados and bananas), reach their best eating quality when allowed to ripen on the tree or plant. However, some fruits are usually picked mature but unripe so that they can withstand the post-harvest handling system when shipped long-distance. Bananas, for example, are treated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Treatment of avocados, kiwi fruit, mangos and pears before marketing is increasingly being performed to provide consumers with the choiche of purchasing ready-to-eat, ripe fruits or mature fruits that can be ripened at home. This practice has, in many cases, resulted in increased sales and profits.

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