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 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.  
</p>
</p>
<img style="box-shadow:none; margin-bottom:-1em;"src="http://2013.igem.org/wiki/images/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" />
<img style="box-shadow:none; margin-bottom:-1em;"src="http://2013.igem.org/wiki/images/f/f8/Tn-2013-project_ethylene-Eth_path.jpg" alt="Ethylene pathway" />
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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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<h2>E. coli</h2>
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<h2> ''EFE in E. coli''</h2>
                 <img src="http://2013.igem.org/wiki/images/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts"/><br/>
                 <img src="http://2013.igem.org/wiki/images/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg" alt="E. coli EFE parts"/><br/>
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<h3>1. Toxicity test</h3>
<h3>1. Toxicity test</h3>
<p>
<p>
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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 non-induced sample.
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<h3>2. Ethylene detection</h3>
<h3>2. Ethylene detection</h3>
<p>
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Then, ethylene was detected using a Micro Gas Chromatograph (see the <a href="http://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 (carbon dioxide, oxygen and ethylene).
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Ethylene production was detected using a Micro Gas Chromatograph (see the <a href="http://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 (carbon dioxide, oxygen and ethylene).
</p>
</p>
<img src="http://2013.igem.org/wiki/images/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" />  
<img src="http://2013.igem.org/wiki/images/c/cf/Tn-2013_EFE_chromatogram.jpg" alt="Ethylene chromatogram" />  
<span class="caption">
<span class="caption">
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<b>Fig. 2:</b> chromatogram obtained using an Agilent Micro GC 3000A. A defined volume of culture was growth until his O.D. has reached 0.5. The cells were then induced with 5 mM Arabinose and putted into an ermetically closed vial with a piearciable septum. After 4 hours at 37 &deg;C in thermoshaker, the samples were connected to the micro GC and a measure was taken.  
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<b>Fig. 2:</b> Ethylene production. Cells transformed with BBa_K1065001 were grown in a thermoshaker until an O.D. of 0.5, placed in hermetically closed vial with a rubber septum and induced with 5 mM Arabinose. Ethylene was measured after 4 hours of induction at 37 &deg;C by connecting the vial to a Agilent Micro GC 3000.  
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<p>
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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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To quantify the amount of ethylene produced the peak integral was converted into ppm.
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<img src="http://2013.igem.org/wiki/images/0/00/Tn-2013_kinetic_EFE_plot-2.png" alt="kinetic_EFE_plot" />
<img src="http://2013.igem.org/wiki/images/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> 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 transformed with BBa_K1065001 and induced with Arabinose  at different O.D.600 and cultured in different conditions.
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<p>
<p>
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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 that induction of the culture at O.D.600 equal to 0.8 a.u. caused a 2-fold increase in ethylene production.
</p>  
</p>  
<div class="separator"></div>
<div class="separator"></div>
<h2>
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E. coli - Blue light circuit
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EFE under the control of a Blue light circuit in '''E. coli'''
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After having characterized Ethylene production using BBa_K1065001, we tried to put EFE under the control of a photoinducible circuit and we then tested his behaviour. We assembled the photoinducible circuit exploiting many subparts from different teams (Uppsala2011 and Berkeley 2006). The construct includes an inverter that allows ethylene production only in presence of light.  
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To build our final system we placed EFE under the control of a photoinducible circuit. We assembled the photoinducible circuit exploiting many subparts from different teams (Uppsala2011 and Berkeley 2006). The construct includes an inverter that allows ethylene production only in presence of light.  
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<p>
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We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065XXX). The sample was then exposed to a blue light led (470 nm) while it was connected to the micro GC (see the protocol page for the adopted method).
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We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065XXX). When the culture reached an OD of 0.7, it was placed in a hermetically closed vial and exposed to a blue light led (470 nm) while it was connected to the micro GC (see the protocol page for the adopted method).
</p>
</p>
<img src="http://2013.igem.org/wiki/images/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" />
<img src="http://2013.igem.org/wiki/images/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" />
<span class="caption">
<span class="caption">
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<b>Fig. 4:</b> ethylene production (ppm) over time (min) of cells induced at O.D.600 = 0.7 exposing the vial to a blue led (470 nm).
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<b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue led light over time (min) of cells transformed with BBa_XX.
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<h2>B. subtilis</h2>
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<h2>EFE in B. subtilis</h2>
<img src="http://2013.igem.org/wiki/images/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/>
<img src="http://2013.igem.org/wiki/images/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg"/>

Revision as of 21:41, 18 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.

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

''EFE in E. coli''

E. coli EFE parts

In E. coli, EFE-catalyzed ethylene production was characterized using BBa_K1065001, which is a composed part with EFE under the control of an 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 non-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

Ethylene production 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 (carbon dioxide, oxygen and ethylene).

Ethylene chromatogram Fig. 2: Ethylene production. Cells transformed with BBa_K1065001 were grown in a thermoshaker until an O.D. of 0.5, placed in hermetically closed vial with a rubber septum and induced with 5 mM Arabinose. Ethylene was measured after 4 hours of induction at 37 °C by connecting the vial to a Agilent Micro GC 3000.

To quantify the amount of ethylene produced the peak integral was converted into 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 transformed with BBa_K1065001 and induced with Arabinose at different O.D.600 and cultured in different conditions.

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

EFE under the control of a Blue light circuit in '''E. coli'''

e.coli_bluelight-EFE_parts

To build our final system we placed EFE under the control of a photoinducible circuit. We assembled the photoinducible circuit exploiting many subparts from different teams (Uppsala2011 and Berkeley 2006). The construct includes an inverter that allows ethylene production only in presence of light.

Photoinduced ethylene production - kinetic assay

We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065XXX). When the culture reached an OD of 0.7, it was placed in a hermetically closed vial and exposed to a blue light led (470 nm) while it was connected to the micro GC (see the protocol page for the adopted method).

EFE-blue_light_plot Fig. 4: Ethylene production (ppm) upon photoinduction with a blue led light over time (min) of cells transformed with BBa_XX.

EFE in B. subtilis

Do you know how plants produce ethylene? Do you know that ethylene is used commercially to ripen some fruits before they enter the market?