Part:BBa_K360100

Promoter + RBS + LuxY + terminator

Vibrio fischeri is a symbiotic marine bacterium which shows luminescence under certain conditions. The light emission reaction is catalyzed by a bacterial luciferase coded in the luxA and luxB genes, both placed within lux operon, this operon also contains genes for the synthesis of the substrates required in this reaction (luxC, luxD, luxE). This is the reaction catalyzed by Vibrio fischeri luciferase (luxAB genes).

FMNH2 + O2 + RCHO -> hv + FMN + H2O + RCOOH(1)

luxY gene codes YFP, a protein that changes the normal light emission wavelength of Vibrio fischeri from 484nm (blue) to 534nm (yellow). luxY gene was isolated from Vibrio fischeri strain Y-1 (2). The yellow emission has been postulated previously to result from energy transfer from an electronically excited species formed in the bacterial luciferase-catalyzed reaction to a secondary emitter protein (1) called YFP.

YFP works together with V. fischeri luxAB products so it must be placed into the same strain (eg. It can either be cotransformated with a plasmid carrying Vibrio fischeri luxAB system or ligated into the same plasmid) (1). Because YFP is a Vibrio fischeri protein, it only works with Vibrio fischeri luciferases although it might work with luciferases from other bacteria species. It should be kept in mind that YFP only changes the light emission wavelength of luciferases, it does not produce light by itself.

Something else to consider is that YFP only works below 18°C, higher temperatures may interfere with the native conformation of the protein disrupting the light shifting function and the only phenotype observed would be that produced by luxAB light emission(1).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This sequence was synthesized.

Position 162 has been changed from G in the article's original sequence to A in order to delete a EcoRI restriction site, however this is a synonymous mutation(3).

Another stop codon (taa) has been added at the end of the sequence.

References

(1) Daubner, S. C., A. M. Astorga, G. B. Leisman, and T. O. Baldwin. 1987. Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein. Proc. Natl. Acad. Sci. U. S. A.

(2) Eckstein, J. W., K. W. Cho, P. Colepicolo, S. Ghisla, and J. W. Hastings, and T. Wilson. 1990. A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turn-over system: energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1. Proc. Natl. Acad. Sci. USA 87:1466-1470.

(3) Cloning and expression of the luxY gene from Vibrio fischeri strain Y-1 in Escherichia coli and complete amino acid sequence of the yellow fluorescent protein. Thomas O. Baldwin, Mary L. Treat, S. Colette Daubner Biochemistry 1990 29 (23), 5509-5515