Team:UNITN-Trento/Project/Ethylene
From 2013.igem.org
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 intermediate molecule (Goto M., Plant and Cell Physiology 2012, 26: 141-150).
![Ethylene pathway](https://static.igem.org/mediawiki/2013/f/f8/Tn-2013-project_ethylene-Eth_path.jpg)
We characterized this gene in two chassis: E. coli and B. subtilis, using different constructs that we designed.
EFE in E. coli![E. coli EFE parts](https://static.igem.org/mediawiki/2013/9/9b/Tn-2013-project_ethylene-BBa_K1065000.jpg)
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.
Ethylene detectionEthylene production was detected using a Micro Gas Chromatograph (see the protocol page for the adopted methodology, Figure 1). The instrument was calibrated using two different air mixtures with well-defined quantities of each molecule (carbon dioxide, oxygen and ethylene).
![Ethylene chromatogram](https://static.igem.org/mediawiki/2013/c/cf/Tn-2013_EFE_chromatogram.jpg)
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 |
We performed a kinetic assay in order to analyze ethylene production over time (see the protocol page for the adopted method).
![kinetic_EFE_plot](https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png)
Figure 2 shows that induction of the culture at O.D.600 equal to 0.8 caused a 2-fold increase in ethylene production.
Toxicity testA toxicity test was performed inducing EFE expression with 5 mM arabinose (Figure 3). The growth curve was then compared to a non-induced sample.
![Toxicity test plot](https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png)
As expected, induced samples showed a decreased growth rate.
EFE under the control of a Blue light circuit in E. coli![e.coli_bluelight-EFE_parts](https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg)
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 (Uppsala 2011 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 and characterization of the circuit check the blue light page of our wiki.
Photoinduced ethylene production - kinetic assayWe performed a kinetic assay in order to analyze ethylene production over time using (BBa_K1065311, Figure 4). At an O.D. of 0.7, the culture was transferred to an hermetically closed vial and exposed to a blue light LED (470 nm). This airtight vial was also connected to the micro GC (see the protocol page for the adopted method).
![EFE-blue_light_plot](https://static.igem.org/mediawiki/2013/2/28/Blue_light_EFE_kinetic.png)
In order to transform B. subtilis with EFE, we decided to exploit two type of vectors designed by the LMU-Munich 2012 iGEM team: pXyl and pSpac. These two vectors were not functionally active: pXyl had a point mutation resulting in a non-transformable vector, and pSpac had a point mutation in the promoter resulting in a non-inducible but constitutive vector. We received from the LMU-Munich team the corrected and functionally active version of both plasmids (functionality was characterized by them).
![](https://static.igem.org/mediawiki/2013/8/85/Tn-2013-project_ethylene-BBa_K1065001.jpg)
We successfully created and transformed the parts shown above (see B. subtilis page for details) but we did not detect any amount of ethylene with the Micro Gas Chromatograph.
Ethylene diffusion in jarsDifferent fruits need different concentrations of ethylene when threated for triggering the ripening process. When we created our ripen machine device, we also though to do an estimation of the concentration of ethylene that there would be in the jar. To do this, we assumed that there was proportionality between air volume / culture volume ratio adopted. Since in the ethylene detection experiment we used a ratio equal to 4 and detected about 101 &plusmin; ppm, we simply applied a proportion to calculate the expected ethylene concetration in the different jars adopted in the experiments (500 ml, 1000 ml and 1500 ml). To threat fruit, we used 300 ml of culture in a 500 ml beute (so with 200 ml of air to add to the jar volume + 100 ml for the air in the connection).
Jar volume (ml) | Air volume (ml) | Culture volume (ml) | Air/culture volumes ratio | Concentration Expected |
---|---|---|---|---|
500 | 800 | 300 | 2.66 | 150.37 |
1000 | 1300 | 300 | 4.33 | 92.37 |
1500 | 1800 | 300 | 6 | 66.66 |
In a second step, we performed a kinetic assay for ethylene diffusion in the jar (1000 ml and 1500 ml), exploting special caps with two connectors (one for the culture and one for the Micro Gas Chromatograph), taking a measure every 45 min for about 12 h.
![](https://static.igem.org/mediawiki/2013/a/af/Ethylene_diffusion_in_jars.png)
2-Oxoglutarate Oxygenase/Decarboxylase (EFE) is a very powerful enzyme that we successfully characterized. We achieved the following results:
- EFE was expressed under the control of an arabinose inducible promoter in E. coli;
- ethylene was detected at the Micro Gas Chromatograph and a quantitative kinetic curve was registered;
- EFE was then inserted into a photoinducible promoter and preliminary analysis showed ethylene production;
- EFE was inserted in two B. subtilis plasmids under the control of two different inducible promoters. We tried to express EFE and measure Ethylene by GC. However, ethylene was not detected. We are now trying to understand if it is a problem of expression or functionality of the enzyme. Interestingly, induced samples showed a distinct smell of sulfur. The presence of sulfur was confirmed by exposure of the culture to a lead acetate paper strip. One hypothesis could be that B. subtilis is capable of converting rapidly ethylene into other mercapto-compounds;
- successfully demonstrated and quantified the presence of ethylene in the jars;
- our system was successfully exploited to accelerate fruit ripening.
Check how we exploited B. fruity to ripen fruit!
![](https://static.igem.org/mediawiki/2013/a/a9/Tn-2013_fruit_exp_pic.jpg)
![](https://static.igem.org/mediawiki/2013/8/81/Tn-2013-tour-T_AAA_DSC_0076.png)