Abstract
This paper presents a generalisable method for the calibration of fluorescence readings on microplate readers, in order to convert arbitrary fluorescence units into absolute units. FPCountR relies on the generation of bespoke fluorescent protein (FP) calibrants, assays to determine protein concentration and activity, and a corresponding analytical workflow. We systematically characterise the assay protocols for accuracy, sensitivity and simplicity, and describe a novel ‘ECmax’ assay that outperforms the others and even enables accurate calibration without requiring the purification of FPs. To obtain cellular protein concentrations, we consider methods for the conversion of optical density to either cell counts or alternatively to cell volumes, as well as examining how cells can interfere with protein counting via fluorescence quenching, which we quantify and correct for the first time. Calibration across different instruments, disparate filter sets and mismatched gains is demonstrated to yield equivalent results. It also reveals that mCherry absorption at 600nm does not confound cell density measurements unless expressed to over 100,000 proteins per cell. FPCountR is presented as pair of open access tools (protocol and R package) to enable the community to use this method, and ultimately to facilitate the quantitative characterisation of synthetic microbial circuits.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
* Swapped Fig3DE with SF10 to pull the lysate figure into the main figures and allow for a more direct comparison of long vs short protocols. * Citation references changed to Vancouver. * Various other minor changes.
Abbreviations
- BCA
- bicinchoninic acid
- BSA
- bovine serum albumin
- EC
- extinction coefficient
- EDTA
- ethylenediaminetetraacetic acid
- FP
- fluorescent protein
- GFP
- green fluorescent protein
- MEFL
- molecules of equivalent fluorescein
- MEFP
- molecules of equivalent fluorescent protein
- OD
- optical density
- PEMS
- particles of equivalent microspheres
- SDS-PAGE
- sodium dodecyl sulphate polyacrylamide gel electrophoresis
- SEVA
- Standardised European Vector Architecture