Part:BBa_K1065204:Design

Efe+Bba_B0015 in BBa_K823024 (pXyl)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 5996
  Illegal suffix found in sequence at 1224
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 5996
  Illegal SpeI site found at 1225
  Illegal PstI site found at 1239
  Illegal NotI site found at 1232
  Illegal NotI site found at 6002
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 5996
  Illegal BglII site found at 319
  Illegal BamHI site found at 5980
  Illegal XhoI site found at 2234
  Illegal XhoI site found at 3152
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 5996
  Illegal suffix found in sequence at 1225
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 5996
  Illegal XbaI site found at 6011
  Illegal SpeI site found at 1225
  Illegal PstI site found at 1239
  Illegal NgoMIV site found at 17
  Illegal NgoMIV site found at 3116
  Illegal NgoMIV site found at 4255
  Illegal AgeI site found at 1070
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4079
  Illegal SapI.rc site found at 1828
  

Design Notes

The construct has been builded with a standard assembly. We firstly optimized the codons of EFE gene for both B. subtilis and E. coli and then synthesized the gene with an RBS sequence upstream of the start codon. We decided to include in the CDS two restriction sites (NgoMIV and AgeI) in order to allow people to use the Freiburg assembly.

GAATTCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGCACC...

...TCGACCGGTTAATACTAGTAGCGGCCGCTGCAG

Source

The coding sequence of EFE gene was taken from Pseudomonas syringae pv. phaseolicola PK2. We used a CDS optimized for both E. coli and B. subtilis that was synthesized by Genescript. This vector is an igem compatible version of the Bacillus subtilis expression vector pXT.

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.
5. http://www.ncbi.nlm.nih.gov/pubmed/11069659 , Derré ''et al.''