In our luminescent system, we coexpressed the luxCDE genes from Photobacterium Phosphoreum with the frp gene from Vibrio Harveyi and a modified LuxAB gene.

[Plasmid Diagram]

To identify the rate limiting enzyme in the synthesis reaction, we have chosen to create three permutations of these genes for expression. By placing a 2A linker sequence between each gene, we can allow for their stoichiometric expression upon translation.

Some kind of “About 2A sequences” blurb: 2A linkers are peptide sequences that cause the ribosome to skip the formation of a peptide bond between its terminal glycine and proline residues. This allows for the stoichiometric coexpression of multiple proteins from a single strand of mRNA. We used the P2A linker, which has the following sequence: (GSG) A T N F S L L K Q A G D V E E N P G P.

[Diagram of Permutations w/ elaborating caption: Because the 2A linkers allow us to control enzyme stoichiometry, we can express these three genes in a precise 1:1:2 ratio. ]

The frp gene from Vibrio Harveyi allows for the regeneration of FMNH2 for the light-generating reaction. In eukaryotic cells, FMNH2 is not replenished at a rate sufficient for strong, consistent luminescence, necessitating the introduction of an enzyme to facilitate the process.

Instead of using the LuxAB genes native to Photobacterium Phosphoreum, we have opted to use an engineered, monomeric luciferase designed for greater stability and luminescent yield. Though the expression of unmodified luciferase has been shown to be sufficient for light-generation, the engineered luciferase has been shown to increase the reaction’s light output, generating a stronger signal.

All coding sequences have been codon optimized for expression in yeast.

[1] Cui B, Zhang L, Song Y, Wei J, Li C, et al. (2014) Engineering an Enhanced, Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts. PLoS ONE 9(10): e107885. doi:10.1371/journal.pone.0107885
[2] Gupta, R. K., Patterson, S. S., Ripp, S., Simpson, M. L. and Sayler, G. S. (2003), Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research, 4: 305–313. doi: 10.1016/S1567-1356(03)00174-0
[3]Szymczak-Workman AL, Vignali KM, Vignali DA. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harbor Protoc. 2012(2): 199–204. doi: 10.1101/pdb.ip067876