Beyond Fluorescein: Use of Fluorescent Protein Calibrants for Direct and Absolute Quantification of Protein Production in Synthetic Biology

While inter-lab calibration standards are approaching mainstream usage in synthetic biology, such calibrations are not in fact sufficient for absolute protein quantification required for modelling synthetic circuits. Fluorescein-based calibration of plate reader and flow cytometry instruments allows the measurement of green fluorescent protein (GFP) in synthetic cells to graduate from arbitrary units to calibrated units, but retains important caveats. Fluorescein is only a good calibrant for green FPs, leaving other FPs uncalibrated, and only provides conversions to units of brightness, not to molecule numbers.

Ideal assay calibrants in molecular biology consist of the same molecule as the one to be measured – in this case, a purified preparation of the appropriate fluorescent protein. Here we show that by using purified FP calibrants, all protein species in a synthetic circuit can be quantified in absolute terms using no advanced instrumentation. We develop a SEVA (Standardised European Vector Architecture)-based expression vector that allows the high-level production of soluble protein and describe a straightforward and reliable protocol for the purification of micrograms of FP, followed by a calibration that relates fluorescence activity to protein mass.

We validate this protocol by calculating conversion factors for a panel of commonly-used FPs including superfolder GFP, mCherry, mScarlet-I and mTagBFP2 and showing that the calculated values match well with both FP brightness values as well as protein abundance data from other studies. We apply this tool for the comparison of the detection sensitivity of multiple laboratory instruments, and use these calibrations to debug failing synthetic circuit components. We also demonstrate that the suspected bias of the presence of mCherry on OD600 measurements is real, but in practice requires extreme overexpression (~100,000 proteins per cell) to have a meaningful impact on cell density estimates.