Difference between revisions of "Part:BBa K911004"
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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.  
 
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.  
  
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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.
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This part was designed so that the 490nm luciferase output acts as an internal control signal, to which the intensity output of the induced luciferase with the spectral shift can be normalised.
  
 
This contruct has been designed to work in bacillus and E.coli, so all the RBSes are at the bacillus consensus.
 
This contruct has been designed to work in bacillus and E.coli, so all the RBSes are at the bacillus consensus.

Revision as of 17:40, 25 September 2012

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.

This part was designed so that the 490nm luciferase output acts as an internal control signal, to which the intensity output of the induced luciferase with the spectral shift can be normalised.

This contruct has been designed to work in bacillus and E.coli, so all the RBSes are at the bacillus consensus. Users looking for a lux operon optimised for B.subtilus may wish to use the one included within this construct.

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 and characterisation.

We contacted the registry about this and were granted exemption from the pSB1C3 standard for this part.

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]