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

Copeland, C. E.; Heitmeier, C. J.; Doan, K. D.; Lee, S. C.; Kwon, Y.-C.

doi:10.1101/2023.08.24.554711

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bioRxiv · 10.1101/2023.08.24.554711

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

Copeland, C. E.·Heitmeier, C. J.·Doan, K. D.·Lee, S. C.·Kwon, Y.-C.

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Abstract

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.

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Copeland, C. E., Heitmeier, C. J., Doan, K. D., Lee, S. C., Kwon, Y.-C.. 2023-08-24. Expanding the Cell-Free Reporter Protein Toolbox by Employing a Split mNeonGreen System to Reduce Protein Synthesis Workload. https://doi.org/10.1101/2023.08.24.554711

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