Part:BBa_K1065203

Efe+Bba_B0015 in pSpac (BBa_K823026)

2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) from Pseudomonas Siringae pv, that catalyzes Ethylene biosynthesis from 2-oxoglutarate. The enzyme was inserted in the IPTG inducible vector pSPAC constructed by the LMU Munich iGEM team 2012 for expression in Bacillus subtilis. 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. If interested in this part please contact us.

NOTE: We used a modified WORKING version of the pSpac vector that has been sent to us from the LMU Munich 2012 team.

Toxicity and sulfur compounds tests

We measured the optical density of cells induced and non induced:

Cells were induced with 0.5 mM of IPTG. The graphic shows that the induced samples (blue trace) grow slightly slower than the controls (red trace).

Cells were induced with 1% of xylose. The graphic shows that the induced samples (blue trace) grow slightly slower than the controls (red trace).

Ethylene production was tested by Gas Chromatography as we previoulsy did for BBa_K1065001. The experiment was performed from both fresh plates and from dry spores. We did not observe any production of ethylene after 4 hours, nor after overnight induction. At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips. Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip:

Detection of sulfur compounds. B. subtilis 168 transformed with BBa_K1065203 were grown until O.D. 0.9 was reached. At this O.D. one sample was then supplemented with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate (see our wiki for more informations. The induced sample darkened much more than the not induced indicating the presence of sulfur compounds (the part BBa_K1065204 behaved similarly).

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 9491
  Illegal suffix found in sequence at 1224
  
• 12
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  Illegal EcoRI site found at 9491
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• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 9491
  Illegal BglII site found at 319
  Illegal BglII site found at 7085
  Illegal BamHI site found at 2601
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 9491
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• 25
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  Illegal prefix found in sequence at 9491
  Illegal XbaI site found at 9506
  Illegal SpeI site found at 1225
  Illegal PstI site found at 1239
  Illegal NgoMIV site found at 17
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  Illegal AgeI site found at 1070
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• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3972
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  Illegal BsaI.rc site found at 7927
  Illegal SapI site found at 2889
  Illegal SapI.rc site found at 6909