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Team:UNITN-Trento/Project/Ethylene
From 2013.igem.org
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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. | 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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<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" /> | ||
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<span class="caption center"> | <span class="caption center"> | ||
<b>Fig. 3:</b> growth curves of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls. | <b>Fig. 3:</b> growth curves of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> and of controls. | ||
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EFE under the control of a Blue light circuit in <i>E. coli</i> | EFE under the control of a Blue light circuit in <i>E. coli</i> | ||
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<img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts"/> | <img src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg" alt="e.coli_bluelight-EFE_parts"/> | ||
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We performed a kinetic assay in order to analyze ethylene production over time using (<a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>). 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). | We performed a kinetic assay in order to analyze ethylene production over time using (<a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>). 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). | ||
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<img src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /> | <img src="https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png" alt="EFE-blue_light_plot" /> | ||
| + | <img class="photo" src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG" /> | ||
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<span class="caption"> | <span class="caption"> | ||
<b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue led light over time (min) of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>. | <b>Fig. 4:</b> Ethylene production (ppm) upon photoinduction with a blue led light over time (min) of cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>. | ||
Revision as of 09:50, 19 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.
EFE in E. coli
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. 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).
Fig. 1: 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 |
2. 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).
Figure 2 shows that induction of the culture at O.D.600 equal to 0.8 caused a 2-fold increase in ethylene production.
3. 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.
Fig. 3: growth curves of cells transformed with BBa_K1065001 and of controls.
As expected, induced samples showed a decreased growth rate.
EFE under the control of a Blue light circuit in E. coli
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 BBa_K1065311 includes an inverter that allows ethylene production only in presence of light. For more details on the design anc characterization of the circuit check the Blue light page of our wiki.
Photoinduced ethylene production - kinetic assay
We performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065311). 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 in B. subtilis
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