Protocol for the Standardisation of Transcriptional Measurements

Hirst, Christopher D.; Ainsworth, Catherine; Baldwin, Geoff; Kitney, R.I.; Freemont, Paul S.

doi:10.1007/8623_2015_148

Cited by 3 publications

(7 citation statements)

References 24 publications

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“…The question of whether the measured fluorescence of FPs in cells is the equivalent of measured fluorescence of the same number of FPs in vitro is less clear. Some authors have found that cells attenuate (or ‘quench’) fluorescence (Zhang et al ., 2009; Hirst et al ., 2015), but the magnitude of the effect has not been systematically investigated, particularly for modest cell concentrations found in a typical E. coli growth assay. We quantified the quenching properties of E. coli cells on our three FPs by mixing an increasing concentration of non-fluorescent cells with purified FPs, and quantifying the difference in apparent fluorescence with added cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It is well known that cells interfere with fluorescence measurements through autofluorescence. Cellular autofluorescence is known to largely impact GFP quantification accuracy (Lichten et al ., 2014), and is corrected for in FPCountR by normalising to the background fluorescence of control cells at a similar OD (akin to Hirst et al ., 2015; Fedorec et al ., 2020). The ‘quenching’ of apparent FP fluorescence by the presence of cells is more rarely considered (Hirst et al ., 2015).…”

Section: Discussionmentioning

confidence: 99%

“…Certain sources of error cannot be adequately addressed by calibration alone. While some authors note that pH can affect the molecular brightness of certain FPs (Hirst et al ., 2015), it could not be compensated for analytically without user input detailing both the pH response profile of the included FPs and the pH of their cells. Fortunately, since the cellular pH in E. coli is limited between pH 7.2-7.8 (Wilks and Slonczewski, 2007), and even pH-sensitive FPs exhibit only mild (<10%) variation in molecular brightness between pH 7-8 (Kneen et al ., 1998; Roberts et al ., 2016), pH-dependent changes in molecular brightness are unlikely to have a large effect on quantifications.…”

Section: Discussionmentioning

confidence: 99%

“…Cellular autofluorescence is known to largely impact GFP quantification accuracy (32), and is corrected for in FPCountR by normalising to the background fluorescence of control cells at a similar OD (akin to (7, 15)). The ‘quenching’ of apparent FP fluorescence by the presence of cells is more rarely considered (14, 15). We have found that the effect size is comparable for different FPs (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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FPCountR: Absolute protein quantification using fluorescence measurements

Csibra

Stan

2021

Preprint

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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 can also reveal 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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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FPCountR: Absolute protein quantification using fluorescence measurements

Csibra

Stan

2021

Preprint

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Systems and Synthetic Biology in Hydrocarbon Microbiology: Tools

Lorenzo

2015

Springer Protocols Handbooks

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Absolute protein quantification using fluorescence measurements with FPCountR

Csibra

Stan

2022

Nat Commun

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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 an ‘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 600 nm 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.

show abstract

Protocol for the Standardisation of Transcriptional Measurements

Cited by 3 publications

References 24 publications

FPCountR: Absolute protein quantification using fluorescence measurements

FPCountR: Absolute protein quantification using fluorescence measurements

Systems and Synthetic Biology in Hydrocarbon Microbiology: Tools

Absolute protein quantification using fluorescence measurements with FPCountR

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