Team:UNITN-Trento/Project/Ethylene
From 2013.igem.org
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Finally between the European jamboree and the championship we were able to take more measures on this circuit in order to obtain further results. | Finally between the European jamboree and the championship we were able to take more measures on this circuit in order to obtain further results. | ||
- | + | <img class="no-bottom" src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png" alt="311_chromatogram" /> | |
- | <span class="tn-caption | + | <span class="tn-caption"><b>Fig. 6:</b> <i>E. coli</i> NEB10β tranformed with <a href="http://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a> were grown in the dark until O.D. 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours after the induction we took measurements of ethylene produced with the micro GC. As we can see ethylene under blue light exposure is produced. In the dark ethylene is not produced.</span> |
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/> | We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that its behavior was not always consistent. Even this time we observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. <br/> | ||
For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results. | For these reasons we also characterized the same circuit without the inverter (<a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a>) to see if the switch would be sharper and obtain better defined results. | ||
<img class="no-bottom" src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png" alt="309_chromatogram" /> | <img class="no-bottom" src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png" alt="309_chromatogram" /> | ||
- | <span | + | <span class="tn-caption"><b>Fig. 7:</b> <i>E. coli</i> NEB10β transformed with <a href="http://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then splitted and kept under the two different conditions. In the dark we could appreciate ethylene production (micro gc measurements) instead with the blue light on there was no ethylene.</span> |
However note that for both circuits not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all. | However note that for both circuits not every colony behaved correctly and sometimes we saw ethylene in the controls or just no ethylene at all. | ||
- | Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior. | + | Further experiments need to be done in order to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior. |
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EFE was inserted in two <i>B. subtilis</i> 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. | EFE was inserted in two <i>B. subtilis</i> 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 <i>B. subtilis</i> is capable of converting rapidly ethylene into other mercapto-compounds. | |
</p> | </p> | ||
<span class="tn-subtitle">Ethylene diffusion in jars</span> | <span class="tn-subtitle">Ethylene diffusion in jars</span> | ||
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<img src="https://static.igem.org/mediawiki/2013/1/1d/Tn-2013_eth_diff_apparatus.JPG" style="display:inline-block;width:40%;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="photo"/> | <img src="https://static.igem.org/mediawiki/2013/1/1d/Tn-2013_eth_diff_apparatus.JPG" style="display:inline-block;width:40%;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="photo"/> | ||
<img src="https://static.igem.org/mediawiki/2013/a/af/Ethylene_diffusion_in_jars.png" style="display:inline-block;width: 58%;height: 307px;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="plot"/> | <img src="https://static.igem.org/mediawiki/2013/a/af/Ethylene_diffusion_in_jars.png" style="display:inline-block;width: 58%;height: 307px;border:2px solid white;box-shadow:2px 2px 4px #323232;" class="plot"/> | ||
- | <span class="tn-caption"><b>Fig | + | <span class="tn-caption"><b>Fig 8:</b> In the left panel, experimental set-up for kinetic measurement of ethylene diffusion. In the right panel, comparison between detected and expected ethylene values. 300 ml of NEB10β cells transformed with <a href="http://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> were induced at O.D. 0.5 and placed into a 500 ml flasks connected to a jar. The jar was simultaneusly connected to the Agilent A3000 micro GC, and a measurment was taken every 45 min for about 12 h.</span> |
- | <span class="tn-quote center">Interestingly, we | + | <span class="tn-quote center">Interestingly, we underestimated the ethylene level in the jars!</span> |
Revision as of 19:08, 26 October 2013
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)
![](https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png)
![311_chromatogram](https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png)
For these reasons we also characterized the same circuit without the inverter (BBa_1065309) to see if the switch would be sharper and obtain better defined results.
![309_chromatogram](https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.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)
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.
Ethylene diffusion in jarsOur ripening machine device is composed of a jar connected to a flask with induced ethylene-producing culture, where the jar contains the fruit to be ripened. A kinetic assay of ethylene in the atmosphere inside our system (jar, connector and flask) was performed by Micro Gas Chromatography and ethylene diffusion from the culture medium was predicted assuming inverse proportionality between detected ethylene and air/culture volume ratio. The estimated data were compared to the results of the kinetic assay as reported in Table 2.
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![](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 (unfortunately also in the dark control);
- EFE was inserted into B. subtilis expression vectors, unfortunately ethylene was not detected upon expression;
- successfully demonstrated and quantified the presence of ethylene in the jars;
- our system was successfully exploited to accelerate fruit ripening.
We succeeded in producing ethylene with our system! Follow our results to discover how we used it 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)