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

The cell-free system offers potential advantages in biosensor applications, but their limited substrate supply poses a challenge in balancing enough sensing resources to detect low limits of analyte while providing a robust output signal. In this study, we harnessed split versions of fluorescent proteins, particularly split super-folder green fluorescent protein and mNeonGreen, to improve energy efficiency and enhance detection limit in the cell-free system. A comparative analysis of the expression of 1-10 and eleventh segments of beta strands in both whole-cell and cell-free platforms revealed distinct fluorescence patterns. Moreover, integrating SynZip peptide linkers substantially improved complementation, achieving a fluorescence intensity reaching 73.6% of the full-length protein and a 4.8-fold increase in expression compared to the split system without the SynZip peptide linkers. The split protein reporter system can enable energy-efficient sensing of low analyte levels in the cell-free system, broadening the toolbox of cell-free biosensor repertoire.