High-Throughput Microbial Assay for Amino Acid Measurement in Ground Maize Seed Samples Utilizing Auxotrophic E. coli.

Abstract

Amino acids are important nutrients in maize grain used for food and feed. Because all 20 amino acids are required for growth and development, a deficiency in a single essential amino acid limits the utilization of dietary protein. In monogastric animals, 10 amino acids must be supplied by the diet and therefore are considered essential. The remaining amino acids can be made from the 10 essential amino acids. Lysine, tryptophan, and methionine are frequently limiting essential amino acids in grain-based diets. Therefore, increasing levels of limiting essential amino acids in grain is an important objective in crop improvement. Standard chromatographic methods for assessing levels of amino acids in grain are extremely accurate, but very expensive. Here, we present a protocol for high-throughput analysis of amino acids in grains, using microbial assays, conducted in 96 well plates, that can be carried out for a fraction of the cost of the standard chromatographic methods. We use Escherichia coli strains that have mutations in the biosynthetic pathway of the amino acid of interest. These strains are auxotrophic, so their growth is proportional to the amount of a specific amino acid in the media. The level of the amino acid of interest in a corn extract is determined by adding the corn extract to the microbial growth medium and measuring the growth of the culture as turbidity in a 96 well plate reader. This protocol is designed for analysis of methionine, but can be adapted for the analysis of any amino acid, by substitution of an appropriate auxotrophic strain of E. coli.