Journal
PHYSICAL BIOLOGY
Volume 12, Issue 6, Pages -Publisher
IOP PUBLISHING LTD
DOI: 10.1088/1478-3975/12/6/066016
Keywords
fluorescence; flavin; escherichia coli; green fluorescent protein; bacterial growth; HPLC; background correction
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Funding
- Investissements d'Avenir Bio-informatique program, project RESET [ANR-11-BINF-0005]
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The intrinsic green autofluorescence of an Escherichia coli culture has long been overlooked and empirically corrected in green fluorescent protein (GFP) reporter experiments. We show here, by using complementary methods of fluorescence analysis and HPLC, that this autofluorescence, principally arise from the secreted flavins in the external media. The cells secrete roughly 10 times more than what they keep inside. We show next that the secreted flavin fluorescence can be used as a complementary method in measuring the cell concentration particularly when the classical method, based on optical density measure, starts to fail. We also demonstrate that the same external flavins limit the dynamical range of GFP quantification and can lead to a false interpretation of lower global dynamic range of expression than what really happens. In the end we evaluate different autofluorescence correction methods to extract the real GFP signal.
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