Team:UNITN-Trento/Project/Ethylene

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<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A.
<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A.

Revision as of 13:53, 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.

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.

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