Team:UC San Diego/Module

Description

All coding sequences have been codon optimized for expression in the yeast Saccharomyces Cerevisiae. By expressing this system in a simple eukaryotic organism, we pave the way for expression of the lux system in higher level organisms, increasing its utility as an in vivo reporter that does not rely on the addition of a substrate.

To identify the rate-limiting enzyme the the Lux system in yeast, we have expressed three permutations of the LuxCDE genes from Photobacterium Phosphoreum in combination with with the frp gene from Vibrio Harveyi and an “enhanced” LuxAB fusion protein 1,2.

LuxAB and frp are expressed under the HXT7 promoter, while the LuxCDE genes are expressed under the TEF1 promoter. Use of this bidirectional promoter system as opposed to the expression of the genes under a single promoter facilitates a modular approach to plasmid design, allowing us to easily test multiple stoichiometric combinations.
Because of the detailed in vitro characterization of its fatty acid reductase complex structure and function, we chose to use the genes from Photobacterium Phosphoreum. A configuration with a single copy of each gene will be used to establish a baseline level of luminescence. Each other permutation of the LuxCDE construct will involve the overproduction of a different Lux C, D, or E enzyme. By comparing luminescence after overproduction for each enzyme to this baseline level, we easily can identify the rate-limiting enzyme and associated rate-limiting step.

To accurately control the relative amounts of each subunit of the complex present in the cell, the genes in our system are separated from each other by P2A linker sequences. These linkers are peptide sequences that cause the ribosome to skip the formation of a peptide bond between its terminal glycine and proline residues3. As a result, the coded proteins are expressed in precise ratios, allowing for the stoichiometric control of multiple proteins from a single strand of mRNA.


Through the use of 2A linkers 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, an essential cofactor for luciferase activity. Previous attempts to express the lux system in yeast in the absence of FMNH2 resulted in weak, unstable light output, which stabilized and increased after addition of the gene2.

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 yield1. 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. This also minimizes the possibility that LuxAB is rate limiting, masking the rate limiting steps in the CDE reactions.

References

[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

Plasmid Maps