Translational_Unit
EFE

Part:BBa_K1065000

Designed by: Thomas Perli   Group: iGEM13_UNITN-Trento   (2013-06-20)
Revision as of 08:09, 8 August 2013 by Xli (Talk | contribs) (→‎Safety)

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]. The CDS has been optimized for both E.coli and B.subtilis. We decided to include an RBS sequence too. The part has been successfully operated while controlled by AraC-pBAD in pSB1C3 (BBa_K1065000) using E.coli as chassis.
This part was cloned by the iGEM Trento 2013 team for the creation of an aerobically engineered pathway for the control of fruit ripening. Further information about this part and its characterization can be found in the iGEM Trento 2013 wiki.

Please note that this part has a modified Prefix and Suffix compatible to RFC25 (Freiburg Assembly). Check the design section for more information.

Usage and Biology

The enzyme was studied deeply 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 was characterized and expressed 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 inflamable (with a concentration of 4 to 5 thousand ppm Ethylene can be inflamable). However we suggest to manage this part carefully. (See BBa_K1065001 for more details.)

Please head over to K1065001 for documentation on characterization of this Part.

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 .,I SHIDAY, .,T AKIKAWAY,. & 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. Return
  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. Return
  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. Return
  4. Fernando Guerrero, Vero´ nica Carbonell., Matteo Cossu., Danilo Correddu, Patrik R. Jones (2012) Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803. PLoS ONE 7(11): e50470.
    5. Return

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