Team:UNITN-Trento/Project/Ethylene

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 intermediate molecule (Goto M., Plant and Cell Physiology 2012, 26: 141-150).

We characterized this gene in two chassis: E. coli and B. subtilis, using different constructs 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.

Ethylene detection

Ethylene 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).

Fig. 1: Ethylene production. E. coli NEB10β 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 an 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. 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).

Fig. 2: ethylene production (ppm) over time (min) of E. coli NEB10β cells transformed with BBa_K1065001 and induced with 5 mM arabinose at different O.D.600 and cultured in different conditions. The control (not-induced sample) did not show any amount of ethylene.

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 test

A toxicity test was performed inducing EFE expression with 5 mM arabinose (Figure 3). The growth curve was then compared to a non-induced sample.

Fig. 3: growth curves of E. coli NEB10β 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 (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 assay

We 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).

Fig. 4: Ethylene production (ppm) upon photoinduction with a blue LED light over time (min) of E. coli NEB10β cells transformed with BBa_K1065311. When we first obtained these results we were really excited. However, the negative control (dark) showed also some ethylene production. The DNA sequencing also indicated some problems in the construct. We have recently fixed this part, confirmed it by sequencing, and submitted to the registry. We did not have the possibility yet to test it for ethylene production. However the part is able to produce amilCP when photoinduced. Since the blue reporter correctly appeared only in the induced sample, we think that ethylene could be properly detected.

Figure 5: amilCP production upon photoinduction. E. coli NEB10β tranformed with BBa_K1065311 was grown in the dark until O.D. 0.6 was reached. The culture was then splitted in two samples and one of them was exposed to a blue led. The samples were grown overnight and the following morning were pelletted. The image clearly shows how only the photoinduced sample (2) produced amilCP while the control (1) kept in the dark remained white. EFE in B. subtilis

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).

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 jars

Different 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

Table 2:ethylene expected in jars.

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.

Fig 6: In the right panel a kinetic assay for ethylene diffusion in jars. 300 ml of culture where induced at O.D. 0.5 and placed into a 500 ml beute connected to a jar. Moreover the jar was connected to the Agilent A3000 micro GC. Every 45 min a measure was taken for about 12 h. The lines show the expected values. In the left panel, a picture of the experimental set up. Interestingly, we underextimated the ethylene level in the jars! Ethylene experiments - Summary

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!