Development of a rapid method fluorescent biosenser to quantitate bioavailable methionine

Abstract

Nutrition is the key to optimizing performance and growth in animals. Amino acids are a component of nutrition that greatly influence growth and therefore must be at proper levels in the blood at all times. One of these vital amino acids is methionine, which, in the vast majority of animals, is the first limiting amino acid. Problems with both deficiencies and toxicities of methionine demand a precise way of quantitating this sulfur amino acid in feeds. Animal and digestibility assays are the current, costly method to quantitate methionine bioavailability. In addition the variability and time efficacy can be hindered by a variety of artifacts. Escherichia coli and other microorganisms have been used to replicate the current methods and to quantitate available amino acid concentrations in feeds. Naturally found in the gastrointestinal microflora, E. coli was chosen as the foundation of an attempt to develop a microbial-based bioassay. An auxotroph was used to accurately indicate the existence of the targeted amino acid. Ultimately the goal of this research was to develop a rapid, inexpensive in vitro assay using a fluorescent E. coli methionine auxotroph to accurately and consistently measure the amount of digestible methionine that exists in feeds and correlate them with in vivo assays. This involved (1) development of a medium that will eliminate background microflora for the assessment of methionine in protein sources (2) analysis of the effects of methionine hydroxy analog on the methionine bioassay (3) development of an assay from the pre-existing assay so that it can utilize microtiter plates to increase replications with less media and (4) development of a fluorescent methionine auxotroph strain of E. coli for accurate and repeatable methionine assessment. Attaining these objectives will allow for an inexpensive and rapid methionine assessment and ultimately improve the nutritional profiles of feedstuffs.