Defined Growth of Prototrophs: Cultivation and isolation of Escherichia coli and related enteric bacteria under chemically defined conditions.

Auxotroph/Prototroph Screening: Replica plating to identify nutritional requirements and select for prototrophic revertants on minimal medium.

Carbon Source Utilization Assays: Testing glucose-dependent growth, metabolic phenotypes, and catabolite repression in defined media.

Physiology and Gene Regulation Studies: Controlled-nutrient experiments for transcriptional regulation, stress responses, and growth rate analyses.

Adaptive Laboratory Evolution (ALE): Selection of fitness-improving mutations during prolonged growth on minimal glucose.

CFU Enumeration on Minimal Media: Counting colonies of cells capable of growth with glucose as the sole carbon source.

Plasmid and Strain Characterization: Evaluating burden, metabolic costs, and genotype–phenotype links in a defined background.

Transport and Metabolism Studies: Quantifying nutrient uptake, glucose utilization rates, and metabolic flux in minimal conditions.

Phenotypic Comparisons: Contrasting wild-type and mutant strains of Escherichia coli or Salmonella enterica on defined agar.

Contamination Control Checks: Verifying culture purity by limiting growth to organisms capable of minimal-glucose utilization.