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

Abstract

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.

Keywords: Cell-free biosensor; Split green fluorescent protein; SynZip; mNeonGreen.

Figure 1

Split fluorescent protein expression in two protein expression systems. a) Split mNG expression in the CFS and whole-cell culture compared to full-length mNG. b) Split sfGFP expression in the CFS and whole-cell culture compared to full-length sfGFP. Black bar: coexpression of the 1–10 and the 11th segments, Red bar: expression of the 1–10 segment alone, Green bar: full-length protein expression. Values represented as mean ± SD, n = 3, **p < 0.01.

Figure 2

Varying DNA concentration ratio of the two split segments in the CFS. a) Split mNG expression values normalized to full-length mNG supplied at the same concentration as the 1–10 segment plasmid DNA. b) Split sfGFP expression absolute values at varying DNA concentrations. Black bar: 1–10 segment DNA ratio, Red bar: 11th segment DNA ratio. Values represented as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 3

Improvement of split mNG with the addition of SynZip linkers. a) Fluorescent output of the CFS with 1 nM DNA concentration and varying purified mNG 1–10 segment concentrations. b) Fluorescent output of the CFS with 3 nM DNA concentration and varying purified mNG 1–10 segment concentrations. c) Percent decrease in protein expression when 2 μg of purified mNG 1–10 segment is added to the CFS expressing full-length mNG at varying concentrations. d) Split mNG expression in the CFS and whole-cell culture compared to full-length mNG with SynZip linkers. Values represented as mean ± SD, n = 3, **p < 0.01, ****p < 0.0001.