Part:BBa_K911004

Synthesised Ratiometric Luciferase construct in non-standard plasmid

This part was designed as a ratiometric luciferase reporter. The first promoter, hyperSpank, is LacI - repressed and controls the transcription of a (vibrio harveyi) luxA gene that has been fused at the N-terminus to an mOrange gene via a flexible linker. This was described by Dachuan Ke and Shiao-Chun Tu (2011) as having an additional peak in its emission spectrum at 560 nm, whereas the normal peak is at 490 nm. This is terminated by b0015. Downstream, pVEG controls the translation of the entire normal lux operon, which is again terminated by b0015.

The idea is that the normal luciferase output acts as an internal control signal, to which the output of the induced luciferase with the spectral shift can be normalised. We designed this to be compatible with our cheap open-source sensing hardware - see our wiki for more details.

This contruct has been designed to work in bacillus and E.coli, so all the RBSes are at the bacillus consensus.

There are some issues with the stability and function of this part necessitating its submission to the registry in a non-standard backbone (BBa_k911006), see experience for further details.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 3988
  Illegal NheI site found at 7654
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1479
  Illegal BglII site found at 4330
  Illegal BglII site found at 6912
  Illegal BglII site found at 8389
  Illegal BamHI site found at 7593
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6308
  
• 1000
  COMPATIBLE WITH RFC[1000]