Team:UNITN-Trento/Project/Ethylene

From 2013.igem.org

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<h1>Results - Ethylene</h1>
<h1>Results - Ethylene</h1>
<p>
<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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EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyses 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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<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>
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<h2>E.coli</h2>
<h2>E.coli</h2>
<p>
<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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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.
</p>
</p>
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<h3>1.Toxicity test</h3>
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<h3>1. Toxicity test</h3>
<p>
<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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A toxicity test was performed inducing EFE expression with 5 mM Arabinose. The growth curve was then compared to a not-induced sample.
</p>
</p>
<img src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" />
<img src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Toxicity test plot" />
<span class="caption">
<span class="caption">
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<b>Fig. 1:</b> a growth curve of cells transformed with BBa_K1065001.
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<b>Fig. 1:</b> growth curve of cells transformed with BBa_K1065001 and of the controls.
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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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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>
<p>
<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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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.
</p>
</p>
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<img src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png" alt="Ethylene chromatogram" />  
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<img src="" alt="Ethylene chromatogram" />  
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<span class="caption">
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<span class="caption center">
<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A.
<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A.
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<p>
<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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The chromatogram clearly shows the presence of a peak corresponding to ethylene, the peak integral was converted to ppm.
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<h3>3.Kinetic assay for ethylene production</h3>  
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<h3>3. Kinetic assay for ethylene production</h3>  
<p>
<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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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).
</p>
</p>
<img src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" />
<img src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" />
<span class="caption">
<span class="caption">
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<b>Fig. 3:</b> plot rappresenting ethylene production during time.
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<b>Fig. 3:</b> ethylene production (ppm) over time (min) of cultures induced at different O.D.600 and cultured at different environmental conditions.
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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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Figure 3 shows how inducing the culture at O.D.600 equal to 0.8 a.u. caused an increase of more than 2 fold in ethylene production.
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<div class="separator"></div>
<span class="quote">
<span class="quote">
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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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Plants naturally produce ethylene starting from a common amino acid: methionine (MET), which is transformed 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, researchers purified the final enzyme of the pathway: ACC oxidase, which converts ACC to ethylene.
</p>
</p>
<img src="" alt="plants_eth_path" />
<img src="" alt="plants_eth_path" />
<span class="caption">
<span class="caption">
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<strong>Fig. 4:</strong> schematich view of plants ethylene producing pathway.
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<strong>Fig. 4:</strong> schematic view of ethylene producing pathway I (plants).
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<span class="quote">
<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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Do you know that ethylene is used commercially to ripen some fruits before they enter the market?
</span>
</span>
<p>
<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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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 avocado, kiwi fruit, mango and pear fruits before marketing is increasingly being performed 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>
</p>
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Revision as of 15:45, 26 August 2013

Results - Ethylene

EFE (Ethylene Forming Enzyme - 2-Oxoglutarate Oxygenase/Decarboxylase) is our keyplayer in triggering fruit ripening. It catalyses 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.

slider

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 curve of cells transformed with BBa_K1065001 and of the 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.

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 cultures 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 an increase of more than 2 fold 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 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, researchers purified the final enzyme of the pathway: 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 avocado, kiwi fruit, mango and pear fruits before marketing is increasingly being performed 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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