Part:BBa_K1725350

pBAD/araC.luxABG - bright

Part K1725350 is a luxABG gene assembly under the pBAD promoter generated by BioBrick assembly by combining luxA (BBa_K1725200), luxB (BBa_K1725201) and luxG (BBa_K1725205) ribosome binding site (RBS) libraries together. RBS BBa_K1725305, BBa_K1725309 and BBa_K1725302 are upstream of luxA, luxB and luxG genes, respectively. The assembly has been shown to induce high levels of bioluminescence in E. coli in a presence of decanal. [http://2015.igem.org/Team:Glasgow/Description More information] about the part.

Design of the RBS library for luxABG

For the construction of the RBS library, a master sequence based on the RBS B0032 was used. 4 nucleotides within the actual ribosome binding site were randomised giving 32 different B0032-derived RBS variants. The predicted efficiency of each RBS library member was estimated using RBS Library Calculator. Originally, RBS Library was designed for every gene in luxABGCDE operon (BBa_K1725352) in order to optimise bioluminescence in E. coli. 32 different BioBrick RBS variants were incorporated upstream of each of the lux genes by PCR, and different libraries were then combined together by BioBrick assembly. This method could potentially produce over 1 billion different variants of the lux operon. Just for the luxABG assembly, there are over 32000 different variants.

Streaks of E. coli colonies with luxABG RBS library. Top left colony is transformed with K1725350.

Design of the RBS library for the lux operon

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NheI site found at 1780
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal XhoI site found at 1608
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3156
  Illegal SapI site found at 961