Cell barcoding with tandem fluorescent proteins enables high-throughput signaling dynamics analysis.

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Cell barcodes are crucial for various biological experiments, including lineage tracing and single-cell analysis.
• Existing methods lack high diversity, live-cell compatibility, and simplified readout.
• There is a need for improved barcoding strategies to enhance throughput and scalability.

Purpose of the Study:

• To introduce and characterize single chain tandem fluorescent protein (sctFP) barcodes.
• To demonstrate the utility of sctFP barcodes for multiplexed biosensing and cell tracking.
• To provide a robust and scalable cell barcoding solution for diverse applications.

Main Methods:

• Construction of sctFP barcodes by linking different fluorescent proteins (FPs) in single polypeptide chains.
• Utilizing fluorescence signal intensity ratios at different wavelengths for sctFP differentiation.
• Application of sctFP barcodes with genetically encoded fluorescent biosensors.

Main Results:

• sctFP barcodes generated from cnidarian FPs showed reliable differentiation based on spectral signal ratios.
• Prokaryotic FPs were less suitable due to cofactor requirements.
• Demonstrated multiplexed tracking of signaling activities using sctFP-compatible biosensors.
• Enabled simplified imaging and analysis pipelines for high-throughput applications.

Conclusions:

• sctFP barcodes offer a robust, scalable, and versatile method for cell barcoding.
• This strategy enhances multiplexing capabilities for fluorescent biosensors.
• sctFP barcodes are compatible with a wide range of biosensor types and experimental contexts.