Absolute protein quantification using fluorescence measurements with FPCountR

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Accurate quantification of protein concentration is crucial for biological research and synthetic biology applications.
• Current fluorescence measurement methods often lack standardization, yielding arbitrary units that hinder inter-study comparisons.
• The need for robust calibration methods is critical for advancing quantitative biology and synthetic microbial circuit characterization.

Purpose of the Study:

• To develop a generalizable method, FPCountR, for calibrating fluorescence readings on microplate readers.
• To convert arbitrary fluorescence units into absolute units for precise protein quantification.
• To provide open-access tools for the scientific community to adopt this calibration method.

Main Methods:

• Generation of bespoke fluorescent protein (FP) calibrants.
• Development of assays to determine protein concentration and activity.
• Implementation of an analytical workflow including an 'ECmax' assay for accurate calibration.
• Quantification and correction for fluorescence quenching by cells.
• Methods for converting optical density to cell counts or volumes.

Main Results:

• FPCountR enables accurate conversion of arbitrary fluorescence units to absolute units.
• The 'ECmax' assay provides accurate calibration, even without FP purification.
• Fluorescence quenching by cells is quantified and corrected for the first time.
• Calibration is demonstrated to be consistent across different instruments and settings.
• mCherry absorption does not interfere with cell density measurements under typical conditions.

Conclusions:

• FPCountR offers a reliable and generalizable method for fluorescence assay calibration.
• The open-access tools facilitate quantitative characterization of synthetic microbial circuits.
• This work advances the standardization of fluorescence measurements in biological research.