Cell-Free Translation Is More Variable than Transcription

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Cell-free gene expression systems, like the PURE system, allow for controlled RNA synthesis using T7 transcriptional promoters.
• However, achieving precise control over protein synthesis levels in these systems remains a challenge.

Purpose of the Study:

• To investigate methods for better controlling protein synthesis levels in cell-free gene expression.
• To identify factors beyond ribosome binding site (RBS) strength that influence protein expression variability.

Main Methods:

• Systematic investigation of various ribosome binding sites (RBSs) with different strengths.
• Analysis of protein synthesis in cell-free systems (PURE system and E. coli cell extract).
• Assessment of factors such as RBS sequence and coding sequence GC content on protein expression.

Main Results:

• RBS strength significantly affects protein synthesis, but explains less than half of the observed variability.
• Protein expression is influenced by additional factors, including the GC content of the coding sequence.
• Protein synthesis exhibits higher variability compared to RNA synthesis, a complexity observed in both T7 and E. coli RNA polymerase systems.

Conclusions:

• Achieving precise control over protein synthesis in cell-free systems requires considering factors beyond RBS strength.
• The inherent complexity of protein synthesis leads to greater variability than RNA synthesis.
• E. coli RNA polymerase systems demonstrate increased variability compared to T7 RNA polymerase systems, reflecting higher biological complexity.