Expanding the Cell-Free Reporter Protein Toolbox by Employing a Split mNeonGreen System to Reduce Protein Synthesis...

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensor Technology

Background:

• Cell-free systems offer biosensing advantages but face limitations in protein synthesis time, hindering sensitive detection.
• Low analyte concentrations challenge fluorescent signal generation in cell-free biosensors.
• Existing cell-free biosensor modules require optimization for enhanced reporter output.

Purpose of the Study:

• To improve the sensitivity of cell-free biosensors for detecting low analyte concentrations.
• To enhance fluorescent signal generation within limited reaction times in cell-free systems.
• To explore the utility of split fluorescent proteins for robust biosensing applications.

Main Methods:

• Utilized split versions of superfolder green fluorescent protein and mNeonGreen for enhanced reporter unit production.
• Performed comparative analysis of beta-strand segment expression (1-10 and 11th) in whole-cell and cell-free platforms.
• Integrated SynZip peptide linkers to improve protein complementation and fluorescence.

Main Results:

• Split fluorescent protein systems increased reporter units synthesized before reaction cessation.
• Distinct fluorescence patterns were observed between whole-cell and cell-free expression systems.
• SynZip peptide linkers significantly enhanced the complementation efficiency of split fluorescent proteins.
• The split reporter system demonstrated potential for higher output when sensing low analyte levels.

Conclusions:

• Split fluorescent protein systems effectively enhance detection limits in cell-free biosensors.
• Optimized protein complementation via SynZip linkers is crucial for signal amplification.
• This approach broadens the capabilities of cell-free biosensors for sensitive analyte detection.