Generation of a green fluorescent protein gene chromosomal insertion containing Escherichia coli strain for gene induction-based quantification of bioavailable lysine

Abstract

The importance of lysine determination in feed materials is crucial for the feed industry because this amino acid can be limiting in many of the cereal materials used for animal feeds. The bacterial gene induction-based assay developed in this study aimed to measure lysine bioavailability in feeds as an alternative analytical method for animal assays. The advantages of a gene induction-based approach include rapid and quantitative estimation of many samples and results that relate a bacterial response to a biological response observed in animals. A whole-cell biosensor strain was constructed using a fluorescent E. coli strain that has an inducible fluorescent phenotype sensitive to extracellular lysine contents. A genetic fusion that links the promoter of cad operon with a green fluorescent protein encoding gene (gfp) was constructed, and a fluorescent assay was developed. A standard lysine curve (R 2 = 0.95) was generated and used for lysine bioavailability quantification of four feed ingredients (whole egg protein, blood-, soybean-, and meat and bone meal). Quantities as low as 50 lg/ml protein of digested samples were sufficient for analyses using the biosensor, except for meat and bone meal. Because of the low levels of free lysine in non-digested samples, fluorescence of these protein sources containing lower than 500 lg/ml protein was not detected (except for soybean meal). The results using enzymatically digested protein sources showed that the test strain emitted a fluorescent response that was proportional to the level of lysine present in the feed samples.

A cad-gfpmut3 plasmid construct in Escherichia coli for gene induction-based quantification of lysine in acid hydrolysates of feedstuffs

“…The overall data indicate that the lysine inducible E. coli strain can be used as an alternative method for the quantification of lysine in acid hydrolysates of feed ingredients. In contrast to a previously developed induction assay for enzymatically derived protein lysine (Zabala‐Díaz et al. 2007), the generation of a plasmid‐borne construct and a gfp mutant with enhanced fluorescence resulted in a stronger fluorescent signal and minimization of background fluorescence interference.…”