Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable...

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Mammalian transcriptional elements exhibit context-dependency, complicating quantitative analysis of multigene expression stoichiometry.
• Understanding and controlling gene expression stoichiometry is vital for various biological functions and applications.

Purpose of the Study:

• To develop a host- and local DNA context-independent transcription system for fine-tuning single and multiple gene expression with predictable stoichiometries.
• To enable precise, dose-dependent control over protein production for advanced applications.

Main Methods:

• A mammalian transcription system was created using modular, programmable bacteriophage promoters and a cognate RNA polymerase (RNAP) fused to a capping enzyme.
• Relative gene expression was controlled by RNAP binding affinity to promoters.
• Multigene expression stoichiometry was predicted using a biochemical model accounting for resource competition.

Main Results:

• The system allowed for gradual fine-tuning of single and multiple gene expression with predictable outcomes.
• Expression of three influenza A virus-like particle (VLP) components was predictably tuned.
• Optimized stoichiometry resulted in a 2-fold increase in intact VLP complex yield.

Conclusions:

• The developed host-independent orthogonal transcription system offers a platform for dose-dependent control of multiple protein expression.
• This system has potential applications in advanced vaccine engineering, cell-fate programming, and other therapeutic areas.