Astbury, M. J.; Schiavon Osorio, A. A.; Victoria, A. J.; McCormick, A. J.

Operons are gene clusters controlled by a single promoter that enable coordinated translation from a single messenger RNA. Here we describe an expansion of the CyanoGate MoClo toolkit to assemble synthetic operons. The versatile CyanOperon system includes two Level 0 acceptor vectors for building interchangeable promoter-ribosome bind site (RBS) combinations and 15 Level 1 acceptor vectors for the hierarchical assembly and expression of up to six genes within a single operon. The system also allows for operon assembly into a self-replicating vector or for chromosomal integration by homologous recombination. To showcase CyanOperon, we assembled the violacein biosynthesis pathway as an operon and demonstrated violacein production in Escherichia coli. We then constructed a 20-part RBS library to examine how spacer length between the Shine-Dalgarno sequence and start codon affects translation in E. coli and the model cyanobacterium Synechocystis sp. PCC 6803. Lastly, we compared the expression of up to three operonic fluorescent markers following chromosomal integration or from a self-replicating vector in E. coli and Synechocystis sp. PCC 6803. The CyanOperon system is publicly available and can be readily integrated with other MoClo systems to accelerate the development of standardized operon assemblies.