Difference between revisions of "Part:BBa K911004"

 
 
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<partinfo>BBa_K911004 short</partinfo>
 
<partinfo>BBa_K911004 short</partinfo>
  
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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 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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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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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. The luxA which does not produce a spectral shift will compete with the luxA which does for binding to luxB, and thus a induction-dependent ratio of 490nm to 560nm intensity produced.
  
 
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.
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Users looking for a lux operon optimised for B.subtilus may wish to use the one included within this construct, which is functional (see experience).
  
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During and after assembly of this sequence, unexpected toxicity issues were observed. This necessitated its assembly in a low copy number vector. This part is submitted to the registry as is, in the suitable backbone provided by the synthesis company. We strongly recommend that you do not attempt to assemble it in psB1C3, as it will kill your cells.
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There are some issues with the stability and function of this as a whole 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.
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The toxicity of the construct causes strong selective pressure against it, and characterisation has been hampered by the tendency of cells to lose parts of the insert. We suspect that the repeated terminator may facilitate recombination, and another team might investigate whether replacing the second terminator aids stability.
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The construct is not behaving entirely as expected, as the e.coli colonies are initially orange, despite mOrange being lacI-repressed. This is probably due to leakage, as the RBSes are very strong in both e.coli and bacillus.
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Orange fluorescence co-segregates with luminescence: colonies that lose their orange colour also lose luminescence (colonies are constitutively luminescent)
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We ran out of time before attempting to insert this into bacillus. Bacillus homology regions would need to be added, but on the upside, the much lower copy number would likely counteract the toxicity issues.
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Further characterisation to follow
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 18:13, 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. The luxA which does not produce a spectral shift will compete with the luxA which does for binding to luxB, and thus a induction-dependent ratio of 490nm to 560nm intensity produced.

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, which is functional (see experience).

There are some issues with the stability and function of this as a whole 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]