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Research

Biological systems - including the simplest cells - exhibit a broad range of functions to thrive in their environment. Research in the Imperial College Centre for Synthetic Biology is focused on the possibility of engineering the underlying biochemical processes to solve many of the challenges facing society, from healthcare to sustainable energy. In particular, we model, analyse, design and build biological and biochemical systems in living cells and/or in cell extracts, both exploring and enhancing the engineering potential of biology.

As part of our research we develop novel methods to accelerate the celebrated Design-Build-Test-Learn synthetic biology cycle. As such research in the Centre for Synthetic Biology highly multi- and interdisciplinary covering computational modelling and machine learning approaches; automated platform development and genetic circuit engineering ; multi-cellular and multi-organismal interactions, including gene drive and genome engineering; metabolic engineering; in vitro/cell-free synthetic biology; engineered phages and directed evolution; and biomimetics, biomaterials and biological engineering.

Publications

Citation

BibTex format

@article{Csibra:2022:10.1038/s41467-022-34232-6,
author = {Csibra, E and Stan, G-B},
doi = {10.1038/s41467-022-34232-6},
journal = {Nature Communications},
title = {Absolute protein quantification using fluorescence measurements with FPCountR},
url = {http://dx.doi.org/10.1038/s41467-022-34232-6},
volume = {40},
year = {2022}
}

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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - 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.
AU  - Csibra,E
AU  - Stan,G-B
DO  - 10.1038/s41467-022-34232-6
PY  - 2022///
SN  - 2041-1723
TI  - Absolute protein quantification using fluorescence measurements with FPCountR
T2  - Nature Communications
UR  - http://dx.doi.org/10.1038/s41467-022-34232-6
UR  - http://hdl.handle.net/10044/1/100789
VL  - 40
ER  -

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