To achieve optimal expression of the Lux operon in E.coli, we had the operon re-synthesized after optimising the usage of codons. This conserves the sequence of amino acids in the gene products, but improves the rate of translation, as more common tRNAs are recruited. Changing the coding DNA sequence also meant changing the curvature of the DNA, which affects the binding affinity of H-NS proteins.

Improved translational speed

Starting with the DNA sequence of the Vibrio fischeri lux operon found on the NCBI database, we used a number of tools to replace the codons used with the most common codons found in the E.coli genome.

Differential Expression

In Vibrio fischeri, LuxA and B are expressed at five times the levels of LuxC, D, E and G. Since all these genes are transcribed on the same mRNA, but have their own Ribosome Binding Sites, this is probably due to differences in codon usage. A number of rare codons are found in Lux C, D, E and G, but not in LuxA and B. Since we did not receive the newly synthesised LuxA and B in time, we constructed a new operon using our LuxC, D, E and G, and the LuxAB BioBrick that was put into the registry by the Edinburgh iGEM team 2009 (BBa_216008). These genes originate from Xenorhabdus luminescens. Compared to Vibrio fischeri, there is only limited amino acid identity in the Lux A and B genes (52% and 66% respectively).