Development of rapid microbial methods for lysine quantification in feed ingredients based on green fluorescent protein fluorescence

Lysine is one of the more limiting amino acids in protein sources for chickens. Since lysine is also an essential amino acid for animals, it is an important component of animal dietary formulation. Therefore, an accurate pre-determination of bioavailable lysine in feedstuffs is important. An optical density (OD) based microbiological assay for lysine determination using E. coli lysine auxotroph has been previously developed. However, because the assay is based on bacterial growth response to extracellular lysine measured as OD, it can be relatively time consuming (10-12h). Therefore, more rapid assays are needed if pre-formulation estimates are required. In this dissertation whole cell fluorescent biosensors for the quantification of bioavailable and total lysine in feed protein sources were developed. The biosensor for quantification of bioavailable lysine was based on the growth response of E. coli to an external source of lysine and lysinecontaining small peptides. Green fluorescent protein (GFP) was inserted in the genome of E. coli lysine auxotroph as a part of a mini-Tn5- transposon by conjugation. Bacterial growth response to external lysine and small peptides was monitored and recorded by measuring the fluorescence emitted by GFP. The second type biosensor developed was designed for the quantification of total lysine. It was based on the measurement of a promoter activity, which was induced and modulated by extracellular concentration of lysine. Cad promoter was amplified from E. coli K-12 genome and was cloned into promoterless gfp plasmid. The construct was electroporated into electrocompetent E. coli cells. The promoter activity was induced under the conditions of low pH and graded concentrations of lysine. Lysine-dose response was measured by the fluorescence of GFP. Both methods were characterized as having a high potential for practical application.