Part:BBa_K1065002

EFE + Terminators

2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyzes Ethylene biosynthesis from 2-oxoglutarate. This enzyme was first 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 a 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 research groups. It was purified and characterized using 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 is inflammable at a concentration ranging from 2.7% to 36%. We characterized this part under the control of an AraC-pBAD promoter. With an air volume/culture volume ratio of 4, about 200 ppm (0.02%) of Ethylene was detected by gas chromatography. This concentration is thus much lower than the inflammability threshold of Ethylene. However, we suggest to use this part carefully and to make all manipulations of open cultures under a chemical hood. (See BBa_K1065001 for more details.)

Characterization

This part was characterized with two different inducible systems in 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 in Neb10β cells

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 an OD of 0.6. The cultures were then split in four samples of equal volume, and two samples were induced with 5 mM Arabinose. Induced samples show a slowed growth rate. This was expected as 5mM arabinose is considered a strong induction that causes moderate stress on cells.

Ethylene detection through Micro Gas-Chromatography

Figure 2 Ethylene detection using an Agilent 3000 Micro GC set up with a plot U column. Neb10β cells were grown until an OD of 0.5. The culture was then split in two samples of equal volume (3 ml) and placed into an hermetically closed vial with a septum and a rubber cap. One of the two samples was induced with 5 mM arabinose. The vials were kept at 37°C under shaking for 4 hours. The vials were then connected to the Micro GC and measurements were performed. Panel A: induced 1.5 ml sample (green curve) and 3 ml sample (red curve) showed a characteristic peak corresponding to Ethylene. On the other hand, the 3 ml non-induced sample (blue curve) didn't show the Ethylene peak. Ethylene concentration 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 in agitation on a thermostatic block at 37 °C until an OD of 0.5 - 0.8 and then connected to the Micro GC. Every 45-60 min a measure was taken for a total experiment time of about 8 hours. Samples were induced at two different OD and this had a marked effect on the amount of Ethylene produced. However, it seems that the Ethylene concentration in the air volume reached saturation after only two hours. The red dashed line indicates the amount of Ethylene detected with a culture left in the shaking incubator for the entire duration of the experiment and subjected to only one measurement. As expected, an higher concentration of Ethylene was measured due to the minimal gas loss using this approach.

Acceleration of fruit ripening

This device was used for accelerating fruit ripening. Many types 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 applied to the acceleration of fruit ripening. We designed hermetically closed jars 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 non-induced) culture at an OD of 0.8. The flasks contained cultures were maintained at 37°C using a laboratory heating plate with a magnetic stirrer. During four to six days, cultures were substituted on a daily basis with a new induced (or non-induced) culture. Furthermore, non-modified jars (i.e. with no connector) were used for the negative control fruit samples (no cells sample). Panel B: ripening of plums. Plums exposed to Ethylene show a more advanced stage of ripening after 4 days of treatment respect to the negative controls (no cells and non- induced BBa_K1065001).

EFE characterization with a photoinducble circuit

This part was also added at the end of a blue light circuit that was built by the UNITN-Trento 2013 team (BBa_K1065310). We preliminarily characterized this part (BBa_K1065311) in E. coli using cells NEB10β cells.

Figure 5 Neb10β cells transformed with BBa_K1065311: pellets after induction time. Cultures at an OD of 0.7 were exposed to a blue LED (470 nm) for about 10 hours (2). Control sample (1) was covered with an aluminum foil and maintained in complete darkness. A substantial difference between pellets' coloration can be observed. AmilCP production probably indicates that also Ethylene would be produced.

We are in the process of characterizing this part by gas chromatography in order to test light-dependent ethylene production. Up to now, we reported amilCP production upon blue light exposure. Given that the blue reporter appeared only in the induced sample, we think that Ethylene would be properly produced.

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.