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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetic Engineering

Background:

• Consistent gene expression is crucial for predictable genetic circuits.
• Previous methods for context-independent translation relied on bicistronic design translational control elements (BCDs).

Purpose of the Study:

• To develop a series of BCDs with tunable strengths for context-independent translation.
• To investigate design features influencing BCD performance.
• To create a versatile expression control cassette for synthetic biology applications.

Main Methods:

• Developed and characterized a series of BCDs with varying strengths.
• Assessed BCD performance across diverse sequence contexts and ligation systems.
• Investigated the impact of start/stop codon spacing and upstream nucleotide identity on translation.
• Engineered BCDs for use in *Rhodococcus* species.

Main Results:

• Created BCDs with expression strengths spanning several orders of magnitude.
• Demonstrated consistent expression levels irrespective of sequence context.
• Showed BCDs are agnostic to common modular cloning ligation sequences.
• Identified key design features affecting leader peptide translation.
• Developed robust BCDs for *Rhodococcus* species.

Conclusions:

• The developed BCDs provide robust, context-independent gene expression control.
• This architecture serves as a generic modular expression control cassette for synthetic biology.
• The BCDs offer flexibility and predictability for genetic circuit design.