Part:BBa_M36719

medium-chain alkane hydroxylating device

We have isolated four genes from the literature which work in tandem to create an alkane-hydroxylating system on substrates of medium chain length (C4-C12). We have rewritten the genetic code for this system using only these four genes, an inducible promoter and strong bicistronic UTR at the start of the entire transcript, and a ribosome binding site before the start of the other genes. alkB1, rubA1, and rubA2 are from Pseudomonas aeruginosa PAO1; rubB is from Pseudomonas fluorescens Pf-5.

This system works by producing the components necessary for the initial terminal oxidation of the alkane substrate to 1-alkanol. AlkB1 makes alkane-1-monoxygenase, which is an enzyme that catalyzes the eaction below. rubA1 and rubA2 make rubredoxins, which are low molecular weight proteins that perform one-electron transfer processes. Rubredoxin reductase (rubB) catalyzes the conversion of reduced rubredoxin to oxidized rubredoxin.

alkane + reduced rubredoxin + O2 ==> 1-alkanol + oxidized rubredoxin + H2O

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 2235
  Illegal PstI site found at 2617
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 2235
  Illegal PstI site found at 2617
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 444
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 2235
  Illegal PstI site found at 2617
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 2235
  Illegal PstI site found at 2617
  Illegal NgoMIV site found at 533
  Illegal NgoMIV site found at 1216
  Illegal NgoMIV site found at 2025
  Illegal AgeI site found at 1613
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2303
  Illegal BsaI site found at 2445