Difference between revisions of "Part:BBa K216008"

Latest revision as of 14:55, 21 October 2009

bacterial luciferase LuxAB of Xenorhabdus luminescens

This part includes the coding sequences of luxA and luxB of Xenorhabdus luminescens, with their native ribosome binding sites. The dimeric bacterial luciferase LuxAB produces light in the presence of oxygen, FMNH2, and a long chain aldehyde. The aldehyde can be provided by the luxCDE gene products, but if these are not present, n-decanal can be added to cultures.

Usage and Biology

Bacterial luciferase, LuxAB, is a heterodimeric flavoprotein which produces blue light (around 495 nm) through oxidation of a long chain aldehyde (tetradecanal in vivo, but n-decanal is usually used in cases where a substrate needs to be added exogenously)(Xi et al, 1991). In vivo, the aldehyde is synthesised/regenerated by the LuxCDE gene products. Bacterial luciferase also requires FMNH2, which is provided by an FMN reductase such as LuxG. The normal structure of the operon is luxCDABEG in most bioluminescent bacteria. For use as a reporter system in heterologous hosts such as Escherichia coli, either luxAB alone may be used (in which case decanal must be provided as substrate), or luxCDABE can be used, in which case the organism can synthesise aldehyde itself. It is not necessary to add luxG as E. coli apparently has sufficient endogenous FMN reductase activity to provide this. Based on kinetics, there are two classes of bacterial luciferase; the 'fast decay' type found in Photobacterium fischeri and P. phosphoreum, and the 'slow decay' type found in Vibrio harveyi and Xenorhabdus luminescens (Valkova et al, 1999). This BioBrick is the luxAB genes encoding luciferase of Xenorhabdus (Photorhabdus) luminescens, with native ribosome binding sites. This enzyme is a favourite choice for biosensors, since X. luminescens (unlike all other known bioluminescent bacteria) is a member of the Enterobacteriaceae, hence a close relative of E. coli, and its luxAB has good codon usage for E. coli. Also, X. luminescens is terrestrial rather than marine, and its luciferase is more thermostable than those from marine bacteria (Westerlund-Karlsson et al, 2002). It also provides a longer-lasting luminescent output (Mitchell et al, 2005).

Comparison to other luminescent reporter systems: the quantum yield of bacterial luciferase is much lower than that of firefly or Renilla luciferase, meaning that the luminescence is much fainter. However, decanal is extremely cheap compared to the D-luciferin and coelenterazine required by these enzymes, and if luxCDE are provided, the organism can produce its own substrate. Thus bacterial luciferase is a good choice for environmental applications.

The X. luminescens luxCDABE operon was previously deposited as BBa_J32007 by Duke 2006 iGEM team, but the Registry sequence analysis shows that the sequence of this part is incorrect (it appears to be a section of E. coli genomic DNA in the ompA region). Thus this part is a replacement for this (together with the luxCDE part K216017, under construction). So far as we can tell, this is the first working version of a bacterial luciferase to be deposited in the Registry.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 530
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 1049

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 ===Usage and Biology===
-Bacterial luciferase, LuxAB, is a heterodimeric flavoprotein which produces blue light (around 495 nm) through oxidation of a long chain aldehyde (tetradecanal in vivo, but n-decanal is usually used in cases where a substrate needs to be added exogenously). In vivo, the aldehyde is synthesised/regenerated by the LuxCDE gene products. Bacterial luciferase also requires FMNH2, which is provided by an FMN reductase such as LuxG. The normal structure of the operon is ''luxCDABEG'' in most bioluminescent bacteria. For use as a reporter system in heterologous hosts such as ''Escherichia coli'', either ''luxAB'' alone may be used (in which case decanal must be provided as substrate), or ''luxCDABE'' can be used, in which case the organism can synthesise aldehyde itself. It is not necessary to add ''luxG'' as ''E. coli'' apparently has sufficient endogenous FMN reductase activity to provide this. Based on kinetics, there are two classes of bacterial luciferase; the 'fast decay' type found in ''Photobacterium'', and the 'slow decay' type found in ''Vibrio'' and ''Xenorhabdus''. This BioBrick is the ''luxAB'' genes of ''Xenorhabdus (Photorhabdus) luminescens''. This is a favourite choice for biosensors, since ''X. luminescens'' (unlike all other known bioluminescent bacteria) is a member of the Enterobacteriaceae, hence a close relative of ''E. coli'', and its ''luxAB'' has good codon usage for ''E. coli''. Also, ''X. luminescens'' is terrestrial rather than marine, and its luciferase is said to be more thermostable than those from other sources (though we can't cite a reference for this).
+Bacterial luciferase, LuxAB, is a heterodimeric flavoprotein which produces blue light (around 495 nm) through oxidation of a long chain aldehyde (tetradecanal in vivo, but n-decanal is usually used in cases where a substrate needs to be added exogenously)(Xi et al, 1991). In vivo, the aldehyde is synthesised/regenerated by the LuxCDE gene products. Bacterial luciferase also requires FMNH2, which is provided by an FMN reductase such as LuxG. The normal structure of the operon is ''luxCDABEG'' in most bioluminescent bacteria. For use as a reporter system in heterologous hosts such as ''Escherichia coli'', either ''luxAB'' alone may be used (in which case decanal must be provided as substrate), or ''luxCDABE'' can be used, in which case the organism can synthesise aldehyde itself. It is not necessary to add ''luxG'' as ''E. coli'' apparently has sufficient endogenous FMN reductase activity to provide this. Based on kinetics, there are two classes of bacterial luciferase; the 'fast decay' type found in ''Photobacterium fischeri'' and ''P. phosphoreum'', and the 'slow decay' type found in ''Vibrio harveyi'' and ''Xenorhabdus luminescens'' (Valkova et al, 1999). This BioBrick is the ''luxAB'' genes encoding luciferase of ''Xenorhabdus (Photorhabdus) luminescens'', with native ribosome binding sites. This enzyme is a favourite choice for biosensors, since ''X. luminescens'' (unlike all other known bioluminescent bacteria) is a member of the Enterobacteriaceae, hence a close relative of ''E. coli'', and its ''luxAB'' has good codon usage for ''E. coli''. Also, ''X. luminescens'' is terrestrial rather than marine, and its luciferase is more thermostable than those from marine bacteria (Westerlund-Karlsson et al, 2002). It also provides a longer-lasting luminescent output (Mitchell et al, 2005).
 Comparison to other luminescent reporter systems: the quantum yield of bacterial luciferase is much lower than that of firefly or ''Renilla'' luciferase, meaning that the luminescence is much fainter. However, decanal is extremely cheap compared to the D-luciferin and coelenterazine required by these enzymes, and if ''luxCDE'' are provided, the organism can produce its own substrate. Thus bacterial luciferase is a good choice for environmental applications.
-The ''X. luminescens luxCDABE'' operon was previously deposited as (BBa...) by (...), but the Registry sequence analysis shows that the sequence of this part is incorrect. Thus this part is a replacement for this (together with the ''luxCDE'' part K2160016, under construction).
+The ''X. luminescens luxCDABE'' operon was previously deposited as '''BBa_J32007''' by Duke 2006 iGEM team, but the Registry sequence analysis shows that the sequence of this part is incorrect (it appears to be a section of ''E. coli'' genomic DNA in the ''ompA'' region). Thus this part is a replacement for this (together with the ''luxCDE'' part K216017, under construction). So far as we can tell, this is the first working version of a bacterial luciferase to be deposited in the Registry.
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