Selection of Auxotrophs/Prototrophs: Isolation and screening of amino acid biosynthesis mutants, including proline auxotrophs and complementation using 250 µg/mL proline.

Defined Carbon Source Utilization: Assessment of metabolic ability to grow on 1% glucose in a minimal, defined background (1X M9 salts).

Metabolic Mutant Screening: Differentiation of strains based on nutrient requirements using 1X M9 salts with 0.1 mM CaCl2 and 0.2 mM MgSO4.

Growth and Fitness Assays: Comparative growth rate and fitness measurements on a defined medium solidified with 1.5% agar.

Genetic Complementation Studies: Selection for restoration of prototrophy via plasmid- or allele-based complementation on minimal plates with 1% glucose and proline.

Colony Isolation and Purification: Isolation of single colonies of Escherichia coli and related organisms under defined conditions.

Antibiotic-Based Selection in Defined Medium: Maintenance of recombinant strains (e.g., Escherichia coli) on minimal agar with added antibiotics as required.

Physiology and Nutrient Requirement Teaching Labs: Educational demonstrations of minimal medium growth, thiamine (1.0 mM) dependence, and amino acid supplementation effects.

Metabolic Pathway Characterization: Evaluation of carbon and amino acid pathway gene function on a controlled minimal matrix.

Strain Stability and Storage: Short-term maintenance of defined-genotype strains (e.g., Escherichia coli) to reduce nutrient-driven variability.