QUANTIFYING METHIONINE WITH A GREEN FLUORESCENT ESCHERICHIA COLI METHIONINE AUXOTROPH

Abstract

ABSTRACT Methionine is one of the essential and primary limiting amino acids in animal nutrition. In this study, an Escherichia coli methionine auxotroph bacterial strain that exhibited a linear growth response to methionine concentrations was transformed with a plasmid containing genes encoding for a green fluorescent protein, in order to develop a fluorescent bioassay technique for methionine quantitation. Transformants were grown on arabinose‐supplemental media and kept at 27C. The strain was also tested in supplemented media with increasing methionine concentrations, and growth response (measured as optical density [OD]) was compared with fluorescence. The transformed E. coli methionine auxotroph exhibited low OD values, but the OD and fluorescent standard curves used for estimating methionine concentrations were linear with increasing increments of methionine concentration (OD, R2 = 0.97 and 0.89; fluorescent, R2 = 0.90 and 0.89) as were the corresponding fluorescent responses (OD versus fluorescent, R2 = 0.85 and 0.91). The transformed fluorescent strain also responded to enzymatic digests of selected animal feed proteins, yielding differences in available methionine among the different feed protein sources. Using the fluorescence‐based bacterial growth assay to quantify methionine in microtiter plates has potential for assessment of methionine levels in animal feed samples.