Revision as of 13:48, 16 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.

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.

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).

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.

[http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Project/Ethylene&action=edit Edit this page] | Main Page

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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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-		<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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-			<strong>Fig. 4:</strong> schematic view of ethylene producing pathway I (plants).
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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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