Interrogating the Function of Bicistronic Translational Control Elements to Improve Consistency of Gene Expression

Context independent gene expression is required for geneticcircuitsto maintain consistent and predicable behavior. Previous efforts todevelop context independent translation have leveraged the helicaseactivity of translating ribosomes via bicistronic design translationalcontrol elements (BCDs) located within an efficiently translated leaderpeptide. We have developed a series of bicistronic translational controlelements with strengths that span several orders of magnitude, maintainconsistent expression levels across diverse sequence contexts, andare agnostic to common ligation sequences used in modular cloningsystems. We have used this series of BCDs to investigate several featuresof this design, including the spacing of the start and stop codons,the nucleotide identity upstream of the start codon, and factors affectingtranslation of the leader peptide. To demonstrate the flexibilityof this architecture and their value as a generic modular expressioncontrol cassette for synthetic biology, we have developed a set ofrobust BCDs for use in several Rhodococcus species.