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).

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 putted 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 characterized this part (BBa_K1065311) in E. coli using cells NEB10b and have preliminary results. We are in the process of acquiring gas-chromatocgraohic measurements in order to test light dependent EFE production. Up to now, we were only able to observe amilCP production upon blue light illumination: since the blue reporter correctly appeared only in the induced control, we think that ethylene could be properly produced.

Figure 5 Neb10β cells transformed with BBa_K1065311: pellets after induction time. We induced cultures when the O.D. reached 0.7 for about 10 hours with a blue LED (2), while the control (1) was being covered with aluminum foil. We can notice a substantial difference between pelletts' colors. AmilCP production probably reflects ethylene synthesis (not measured yet). We are in the process of characterizing this part more properly and getting some gas-chromatographic.

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.