Part:BBa_K1065000

2-oxoglutarate oxygenase/decarboxylase (EFE)

2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyze Ethylene biosynthesis from 2-oxoglutarate. This enzyme was firstly purified from Pseudomonas Siringae pv. phaseolicola PK2, a 2-oxoglutarate-dependent ethylene producing bacterium [1].
This part was cloned by the iGEM Trento 2013 team for the creation of an aerobically engineered pathway for the control of fruit ripening. The part has been successfully operated while controlled by AraC-pBAD in pSB1C3 (BBa_K1065001) using E.coli as chassis. Further information about this part and its characterization can be found in the iGEM Trento 2013 wiki.

Usage and Biology

The enzyme was thoroughly studied by many reasearch groups. It was purified and characterized with an in vitro test [2]. It was then transformed and ectopically expressed in E.coli [3] and in Synecocystis sp [4].

Safety

This part produces ethylene, a compound that can be inflammable at a concentration between 2.7 to 36%. We characterized this part under the control of an AraC-pBAD promoter. With a air volume/culture volume ratio = 4, we detected about 200 ppm of Ethylene. This concentration is not dangerous and not inflammable. However we suggest to manage this part carefully. (See BBa_K1065001 for more details.)

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Characterization

This part was characterized with with two different inducible systems in E. coli Neb10β cells:

• under the control of an AraC-pBAD promoter inducible by arabinose (BBa_K1065001)
• under the control of a photoinducible circuit (BBa_K1065311 and BBa_K1065309).

EFE characterization with AraC-pBAD promoter

Figure 1 Effect of EFE on cell growth. Cell density was measured at different time points to determine the effect of EFE expression. Neb10β cells were grown at 37 °C in LB until it was reached an OD of 0.6. The cells were then splitted in four samples of equal volume. Two samples were then induced with 5 mM Arabinose. Induced samples show a slowed growth rate, as espected (5mM arabinose is a strong induction that causes stress on cells). However, cell growth is not completely inhibited so EFE is not highly toxic

Ethylene detection through Micro Gas-Chromatography

Figure 2 Ethylene detection with an Agilent 3000 Micro GC set up with a plot U column. Neb10β cells were grown until O.D.600 reached 0.5. The culture was then splitted in two samples of equal volume (3 ml) and placed into an hermetically closed vial with a septum with a rubber cap. One of the two sample was induced with 5 mM Arabinose. The vials were left in the thermoshaker for 4 hours. After that, the vials were connected to a micro GC and a measure was taken. Panel A: a 1.5 ml sample induced (green curve) and 3 ml sample induced (red curve) showed a characteristic peak corresponding to Ethylene. On the other hand, the 3 ml not induced sample (blue curve) didn't show the peak. Ethylene was estimated to be 61 ± 15 ppm for the 1.5 ml culture and 101 ± 15 ppm for the 3 ml culture. Panel B: picture of the vial connected to the micro GC.

Kinetic assay for Ethylene production

Figure 3 Kinetic assay for Ethylene production. Neb10β cells were grown until an O.D. of 0.5 - 0.8 and then connected to the micro GC, while in agitation on a thermoblock at 37 °C for the entire duration of the experiment. Every 45/60 mins a meausure was taken for a total of about 8 hours. Samples were induced at two differents O.D.600 and this had big effect on the amount of ethylene produced. However, it seems that the Ethylene concentration in the air space reached saturation after only two hours. The red dashed line indicates the amount of ethylene detected with a culture left in the thermoshaker for the all duration of the experiment and subjected to only one measurement. As expected an higher value of ethylene was measured due to the minimal gas loss with this approach.

Acceleration of fruit ripening

This device was used for accelerating fruit ripening. Many type of fruit were tested and ripened successfully. For more information and details please visit the UNITN wiki page .

Figure 4 Acceleration of fruit ripening. Panel A: our system was exploited for the acceleration of fruit ripening. We designed an hermetically closed jar with a rubber hose connector. These jars contained our test-fruit and each one was connected to a flask. The flasks contained 300 ml of induced (or not) culture when O.D.600 reached 0.8. The flasks contained a cultures maintained at 37 °C using a laboratory heating plate while stirring. For four to six days, every morning the culture in the flasks was substituted with a new induced (or not) culture. Furthermore, non-modified jars (i.e.: with no connector) were adopted to contain the negative control fruit samples (no-cells). Panel B: ripening of plums. Plum exposed to ethylene, show a more advanced stage of ripening after 4 days of treatment when compared to two negative controls (no-cells and BBa_K1065001 not induced).

EFE characterization with a photoinducble circuit

We controlled ethylene production with two variants of a photoinducible circuit (with inverter BBa_K1065311 and without inverter BBa_K1065309) in E. coli using cells NEB10β. BBa_K1065311 produces ethylene upon blue light exposure, while BBa_K1065309) produces ethylene in the dark. For a detailed description of the circuits please refer to the corresponding parts page.

Figure 5 Neb10β cells 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, 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 and obtain better defined results.

Figure 6 Neb10β cells transformed with BBa_1065309 were 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 (micro gc measurements) instead in the presence of blue light there was no ethylene produced.

Also in this case not every colony behaved correctly and sometimes we saw ethylene in the control or just no ethylene at all. However the on/off switch was better defined with this part. Further experiments need to be done 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 319
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 17
  Illegal AgeI site found at 1070
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Goto M, Shiday I, Akitaway T, Hyodoh, (1985). Ethylene production by the Kudzu strains of Pseudomonas syringae pv. phaseolicola causing halo blight in Pueraria lobata (Willd) Ohwi. Plant and Cell Physiology 26, 141-150.
2. Nagahama K, Ogawa T, Fujii T, Tazaki M, Tanase S, et al. (1991) Purification and properties of an ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2. Journal of General Microbiology 137: 2281–2286.
3. Fukuda H, Ogawa T, Ishihara K, Fujii T, Nagahama K, et al. (1992) Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2. Biochem Biophys Res Commun 188: 826–832.
4. Guerrero F, Carbonell. V., Cossu M, Correddu D, Jones PR (2012) Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803. PLoS ONE 7(11): e50470.