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 its limited time for protein synthesis poses a challenge in creating enough fluorescent signals to detect low limits of the analyte while providing a robust sensing module at the beginning. In this study, we harnessed split versions of fluorescent proteins, particularly split superfolder green fluorescent protein and mNeonGreen, to increase the number of reporter units made before the reaction ceased and enhance the detection limit in the cell-free system. A comparative analysis of the expression of 1-10 and 11th segments of beta strands in both whole-cell and cell-free platforms revealed distinct fluorescence patterns. Moreover, the integration of SynZip peptide linkers substantially improved complementation. The split protein reporter system could enable higher reporter output when sensing low analyte levels in the cell-free system, broadening the toolbox of the cell-free biosensor repertoire.