Escherichia coli is an important model organism in scientific research. Different strains can carry different genetic properties, and gene knockouts are used to study effects on phenotype. Deletion of the aroE gene in the MG1655 and ATCC 25922 strains of Escherichia coli, coding for an essential enzyme in the biosynthesis pathway for aromatic amino acids, resulted in auxotrophy. Subsequent selection for prototrophy revealed that the auxotrophic phenotype could be suppressed at a frequency of approximately 10-8 in ATCC 25922 but not in MG1655. The aim of this study was to identify the mutation(s) that suppressed the phenotype of the aroE gene deletion and determine why suppression occurred in ATCC 25922, but not in MG1655. Independently selected mutants were analysed by whole genome sequencing, but no obvious genetic alterations were identified. This prompted an evaluation of the phenotypic stability of the selected mutants. Mutants were growth in rich medium then tested for auxotrophy, which revealed that the mutant phenotype was highly unstable. While the initial research question could not be answered, both the frequency of occurrence and instability of the selected mutant phenotype, are important clues. Genetic instability is associated both with gene duplication, for which no evidence was found in the genome sequence, and epigenetic changes, which are not revealed by standard DNA sequencing. In conclusion, an auxotrophy bypass mechanism has been shown to exist in ATCC 25922 with no obvious genotypic source. Further experimentation, including methylome sequencing, might provide more leads.