Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Current cell-free expression systems rely on bacteriophage RNA polymerases, limiting transcription modularity for biological information studies.
• Developing cell-free systems with broader transcription capabilities is crucial for in vitro analysis of complex informational processes.

Purpose of the Study:

• To create an efficient cell-free expression system utilizing the endogenous Escherichia coli (E. coli) RNA polymerase and sigma factor 70.
• To enhance protein production and engineer a more translatable enhanced green fluorescent protein (eGFP) for cell-free applications.

Main Methods:

• Prepared a cell-free expression system using only E. coli's endogenous transcription machinery.
• Optimized plasmid constructs with regulatory elements for increased protein expression.
• Characterized protein production using three adenosine triphosphate (ATP) regeneration systems: creatine phosphate, phosphoenolpyruvate, and 3-phosphoglyceric acid.

Main Results:

• Achieved protein production yields of approximately 0.75 mg/ml for Firefly luciferase and eGFP in batch mode.
• Engineered a novel eGFP variant with improved translatability in cell-free systems.
• Identified 3-phosphoglyceric acid as the optimal ATP regeneration system for maximum protein yield.

Conclusions:

• The developed E. coli-based cell-free system produces active proteins comparable to bacteriophage systems.
• This system offers enhanced possibilities for engineering and studying synthetic gene circuits and biological information processes in vitro.
• Leverages the extensive library of E. coli sigma factor 70-specific promoters and operators for greater regulatory control.