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 vials 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, 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 but then we found that the negative control (dark) also showed some ethylene production. DNA sequencing confirmed that there were some problems with this construct. We have now fixed this part, confirmed it by sequencing, and submitted it to the registry. This updated version of BBa_K1065311 is able to produce amilCP when photoinduced. Importantly, the blue reporter correctly appeared only in the induced sample, so we think that ethylene could be properly produced.

Figure 5: amilCP production upon photoinduction. E. coli NEB10β transformed with BBa_K1065311 were 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. New GC measurements on the circuit: we can control ethylene production in a non-chemical way! Finally between the European jamboree and the world championship we were able to take more measurements on this circuit in order to obtain further results. Fig. 6: E. coli NEB10β transformed with BBa_K1065311 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 from the induction we measured the amount of ethylene produced with the micro GC. Ethylene is produced upon blue light exposure (92 ±15 ppm), while it is not produced in the dark. We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that the behavior of the circuit 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.
For these reasons we also characterized the same circuit without the inverter (BBa_1065309) to see if the switch would be sharper. Fig. 7: E. coli NEB10β transformed with BBa_1065309 was grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (37 ±15 ppm) instead in the presence of blue light there was no ethylene produced. 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 the on/off switch was better defined with the circuit without an inverter. 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. 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

Our 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.

Jar volume (ml)	Air volume in the jar + connections (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: prediction of ethylene diffusion in jars.
Fig 8: 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 BBa_K1065001 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. Interestingly, we underestimated 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 two different versions of a photoinducible circuit (with and without an inverter), GC measurements were taken and the results were consistent with the engineered function;
• 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.