Difference between revisions of "Part:BBa K911004"

Revision as of 13:48, 22 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.

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, 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]

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 __NOTOC__
 <partinfo>BBa_K911004 short</partinfo>
-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.
+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.
-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.
+There are some issues with the stability and function of this part, see experience for further details.
-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.
-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.
-Orange fluorescence co-segregates with luminescence: colonies that lose their orange colour also lose luminescence (colonies are constitutively luminescent)
-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.
-Further characterisation to follow
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