Team:UNITN-Trento/Project/Ethylene

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<h1>Results - Ethylene</h1>
<h1>Results - Ethylene</h1>
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<p>
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EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyses ethylene from 2-Oxoglutarate (a TCA cycle intemediate molecule).
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</p>
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<p>EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyses ethylene from 2-Oxoglutarate (a TCA cycle intemediate molecule). </p>
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<p>
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We characterized that gene in two chassis: <i>E. coli </i>and <i>B. subtilis</i> using different contstructs that we designed.
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<p>We characterized that gene in two chassis: <i>E. coli </i>and <i>B. subtilis</i> using different contstructs that we designed.</p>
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</p>
<img src="" alt="slider">
<img src="" alt="slider">
<h2>E.coli</h2>
<h2>E.coli</h2>
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<p>
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<p>Using <i>E. coli</i> as chassis,  we characterized EFE using BBa_K1065001 part, wich is composed by AraC-pBAD promoter + EFE.</p>
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Using <i>E. coli</i> as chassis,  we characterized EFE using BBa_K1065001 part, wich is composed by AraC-pBAD promoter + EFE.
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</p>
<h3>1.Toxicity test</h3>
<h3>1.Toxicity test</h3>
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<p>
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A toxicity test was done inducing cells to express EFE with 5mM Arabinose. A growth curve was then compared to a not-induced sample.
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</p>
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<p>A toxicity test was done inducing cells to express EFE with 5mM Arabinose. A growth curve was then compared to a not-induced sample.</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">
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<center><img style="width:80%;"src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot"></center>
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<b>Fig. 1:</b> a growth curve of cells transformed with BBa_K1065001.
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<span class="caption"><strong>Fig. 1:</strong> a growth curve of cells transformed with BBa_K1065001. </span>
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</span>
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<p>As expected, induced saples showed a slowed but a not completely inhibited growth rate.</p>
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<p>
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As expected, induced saples showed a slowed but a not completely inhibited growth rate.
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</p>
<h3>2.Ethylene detection</h3>
<h3>2.Ethylene detection</h3>
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<p>
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The next step was to detect ethylene using a Micro Gas Chromatograph (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#ethylene-detection-assay">protocol page</a> for method adopted).
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</p>
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<p>The next step was to detect ethylene using a Micro Gas Chromatograph (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#ethylene-detection-assay">protocol page</a> for method adopted).</p>
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<img style="width=80%;"src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Ethylene chromatogram" />  
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<img style="width=80%;"src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Ethylene chromatogram">  
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<span class="caption">
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<span class="caption"><strong>Fig. 2:</strong> chromatogram obtained using an Agilent Micro GC 3000A.</span>
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<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A.
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<p>The chromatogram clearly shows the presence of a peak corresponding to ethylene. The integral was then converted to ppm thanks to a calibration straight previously done using two different air mixtures (containing well-defined quantity of each molecule).</p>
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</span>
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<p>
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The chromatogram clearly shows the presence of a peak corresponding to ethylene. The integral was then converted to ppm thanks to a calibration straight previously done using two different air mixtures (containing well-defined quantity of each molecule).
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</p>
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<center><table id="ethylene detected">
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<center>
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<tr>
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<table id="ethylene detected">
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<th>Sample</th>
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<tr>
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<th>Ethylene detected</th>
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<th>Sample</th>
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</tr>
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<th>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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<tr>
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<td>0 &plusmn; 15 ppm</td>
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<td>Not induced</td>
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</tr>
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<td>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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<tr>
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<td>61 &plusmn; 15 ppm</td>
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<td>Induced V = 1.5 ml</td>
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</tr>
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<td>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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<tr>
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<td>101 &plusmn; 15 ppm</td>
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<td>Induced V = 3 ml</td>
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<td>101 &plusmn; 15 ppm</td>
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</table></center>
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<span class="caption"><strong>Table. 1:</strong> ethylene detected.</span>
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</table>
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</center>
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<span class="caption">
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<b>Table. 1:</b> ethylene detected.
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</span>
<h3>3.Kinetic assay for ethylene production</h3>  
<h3>3.Kinetic assay for ethylene production</h3>  
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<p>
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In order to analyze the production of ethylene during time, we performed a kinetic assay (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for method adopted).
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</p>
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<p>In order to analyze the production of ethylene during time, we performed a kinetic assay (see the <a href="https://2013.igem.org/Team:UNITN-Trento/Protocols#kinetic-ethylene-production">protocol page</a> for method adopted).</p>
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<img style="width:80%"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 style="width:80%"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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<span class="caption"><strong>Fig. 3:</strong> plot rappresenting ethylene production during time.</span>
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<b>Fig. 3:</b> plot rappresenting ethylene production during time.
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</span>
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<p>As you can see from the plot, inducing culture at an O.D. of 0.8 caused an increase of more than 2 fold in ethylene.</p>  
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<p>
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As you can see from the plot, inducing culture at an O.D. of 0.8 caused an increase of more than 2 fold in ethylene.
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</p>  
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<span class="ita">Do you know how plants produce ethylene?</span>
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<span class="quote">
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Do you know how plants produce ethylene?
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</span>
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<p>Plants naturally produce ethylene starting from a common amino acid, methionine MET, wich is energized by ATP to produce S-adenosyl methionine SAM. The key enzyme in the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid ACC, the immediate precursor of ethylene. Quite recently researches purified the final enzyme in the pathway, ACC oxidase, wich converts ACC to ethylene. </p>
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<p>
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Plants naturally produce ethylene starting from a common amino acid, methionine MET, wich is energized by ATP to produce S-adenosyl methionine SAM. The key enzyme in the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid ACC, the immediate precursor of ethylene. Quite recently researches purified the final enzyme in the pathway, ACC oxidase, wich converts ACC to ethylene.
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</p>
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<img src="" alt="plants_eth_path">
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<img src="" alt="plants_eth_path" />
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<span class="caption"><strong>Fig. 4:</strong> schematich view of plants ethylene producing pathway.</span>
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<span class="caption">
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<strong>Fig. 4:</strong> schematich view of plants ethylene producing pathway.
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</span>
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<span class="ita">Do you know that ethylene is used commercially to ripen some fruits before entering in the market?</span>
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<span class="quote">
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Do you know that ethylene is used commercially to ripen some fruits before entering in the market?
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</span>
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<p>All fruits, with a few exceptions (European pears, avocados and bananas), reach their best easting 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 postharvest handling system when shipped long-distance. Bananas for example are threated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Ripening of avocado, kiwifruit, mango and pear fruits before marketing is increasingly being used to provide consumers with the choiche of purchasing ready-to-eat, ripe fruits or mature fruit that can be ripened at home. This practice has, in many cases, resulted in increased sales and profits.</p>
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<p>
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All fruits, with a few exceptions (European pears, avocados and bananas), reach their best easting 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 postharvest handling system when shipped long-distance. Bananas for example are threated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Ripening of avocado, kiwifruit, mango and pear fruits before marketing is increasingly being used to provide consumers with the choiche of purchasing ready-to-eat, ripe fruits or mature fruit 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 15:19, 26 August 2013

Results - Ethylene

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

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

slider

E.coli

Using E. coli as chassis, we characterized EFE using BBa_K1065001 part, wich is composed by AraC-pBAD promoter + EFE.

1.Toxicity test

A toxicity test was done inducing cells to express EFE with 5mM Arabinose. A growth curve was then compared to a not-induced sample.

Toxicity test plot Fig. 1: a growth curve of cells transformed with BBa_K1065001.

As expected, induced saples showed a slowed but a not completely inhibited growth rate.

2.Ethylene detection

The next step was to detect ethylene using a Micro Gas Chromatograph (see the protocol page for method adopted).

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 integral was then converted to ppm thanks to a calibration straight previously done using two different air mixtures (containing well-defined quantity of each molecule).

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.

3.Kinetic assay for ethylene production

In order to analyze the production of ethylene during time, we performed a kinetic assay (see the protocol page for method adopted).

kinetic_EFE_plot Fig. 3: plot rappresenting ethylene production during time.

As you can see from the plot, inducing culture at an O.D. of 0.8 caused an increase of more than 2 fold in ethylene.

Do you know how plants produce ethylene?

Plants naturally produce ethylene starting from a common amino acid, methionine MET, wich is energized by ATP to produce S-adenosyl methionine SAM. The key enzyme in the pathway, ACC synthase, converts SAM to 1-aminocycloprane-1-carboxylic acid ACC, the immediate precursor of ethylene. Quite recently researches purified the final enzyme in the pathway, ACC oxidase, wich converts ACC to ethylene.

plants_eth_path Fig. 4: schematich view of plants ethylene producing pathway. Do you know that ethylene is used commercially to ripen some fruits before entering in the market?

All fruits, with a few exceptions (European pears, avocados and bananas), reach their best easting 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 postharvest handling system when shipped long-distance. Bananas for example are threated with 10-100 ppm of ethylene to initiate their ripening during transport or at destination handling facilities. Ripening of avocado, kiwifruit, mango and pear fruits before marketing is increasingly being used to provide consumers with the choiche of purchasing ready-to-eat, ripe fruits or mature fruit that can be ripened at home. This practice has, in many cases, resulted in increased sales and profits.

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