- BBa_K1725350: 721 I0500-K1725305-luxA-K1725309-luxB-K1725302-luxG
- BBa_K1725351: K1725080-K1725307-luxC-K1725302-luxD-K1725301-luxE
- BBa_K1725352: I0500-K1725305-luxA-K1725309-luxB-K1725302-luxG-K1725080-RBS-luxC-RBS-luxD-RBS-luxE
Ribosome Binding Site Library Summary
To optimise bioluminescence in Escherichia coli by creating a range of Ribosome Binding Sites (RBS) for each of the six genes in the luxCDABEG operon from Aliivibrio fisheri, originally submitted to the registry as a single BioBrick (K325909) in 2010 by the Cambridge team.
Basic Parts: RBS library
For the Bioluminescence part of our project we used the luxCDABEG operon from A. fischeri introduced to iGEM by the Cambridge team in 2010. Five lux genes are known to be essential for bioluminescence production: luxA and luxB encoding bacterial luciferase and luxC, luxD and luxE encoding an enzyme complex that synthesises tetradecanal, a substrate for the luciferase. A sixth gene, luxG encodes a flavin reductase that provides reduced flavin mononucleotide for the bioluminescence reaction resulting in an enhanced light output. Initially we decided to optimise bioluminescence in E. coli by rearranging the whole Lux operon and placing a defined relatively-weak ribosome binding site – B0032 – upstream of each of the six lux genes, as described on our Bioluminescence page. Taking this approach further, we thought of adjusting bioluminescence in E. coli by creating a B0032-derived Ribosome Binding Site library for each lux gene. The idea behind this was to create a range of RBS combinations in a lux operon and therefore, generate E. coli strains of different bioluminescence intensity. We assumed that the most favourable RBS arrangements in lux operon should be observed in the E. coli colonies emitting the most light.
DesignFor the construction of the RBS library, we used a master sequence based on the RBS B0032 (Figure 1). 4 nucleotides within the actual ribosome binding site were randomised giving 32 different B0032-derived RBS variants. The predicted efficiency of each RBS library member was estimated using RBS Library Calculator (ref: http://msb.embopress.org/content/10/6/731) for every lux gene. Theoretically, with 32 different RBS variants for each of the six lux genes, the final RBS library for the complete lux operon would have over a billion different RBS combinations.
Strategy and approaches
Randomised PCR and Cloning, Cloning, Cloning
Mr. Bright: luxG
The luxCDE story
Comparison to luxCDABEG (BBa_K325909)