LTEE Media Recipes v2
Authors: E Chavarria-Palma Jesus 
Published: July 2023

This protocol describes recipes to prepare growth media and reagents used in the E. coli long-term evolution experiment (LTEE). Section 1: DM-glucose, Davis-Mingioli liquid medium supplemented with glucose Section 2: Sterile Saline Section 3: TA agar, Tetrazolium Arabinose agar Section 4: MG agar, Minimal Glucose agar (equivalent to DM agar) Section 5: MA agar, Minimal Arabinose agar Section 6: MC agar, Minimal Citrate agar Section 7: CC agar, Christensen Citrate agar DM-glucose: Davis-Mingioli medium (or sometimes called Davis Minimal medium) supplemented with glucose is used for propagating the LTEE populations and for performing related experiments. For propagating the LTEE, glucose is added to a concentration of 25 mg/L, which we refer to as "DM25". DM25 supports a stationary-phase density of about 5×107 cells/mL for E. coli REL606 and REL607, the founding strains of the LTEE. (The stationary-phase density of evolved LTEE clones varies, but tends to be approximately half that of the ancestral strains.) DM with higher concentrations of glucose is used for reviving cells from freezer stocks or for growing many cells to harvest for certain experiments. These other DM formulations are named in an analogous fashion of DMX, where X is the concentration of glucose in mg/L (e.g., 1000 mg/L glucose in DM1000). Sterile Saline: Used to dilute E. coli cultures, for instance when plating on agar to isolate colonies or to count CFUs to determine cell titers. TA agar: Tetrazolium Arabinose agar plates are used for distinguishing E. coli cells that can grow on the sugar arabinose (Ara+) from those that cannot (Ara–). Plating dilutions that give 150-250 colonies are used for monitoring the LTEE for contamination and also for co-culture competition assays that measure the relative fitness of two strains. Colonies grown from Ara– cells appear red on TA agar, while those of Ara+ strains appear pinkish-white. These phenotypes are very clear after 24 hours of incubation at 37°C for the REL606 (Ara–) and REL607 (Ara+) ancestors of the LTEE. Colonies of evolved clones can exhibit a wide variation of these color phenotypes. Some evolved clones may take longer than 24 hours to form visible colonies on TA. MG agar: Minimal Glucose agar has the same base composition as DM-glucose liquid medium, except agar is added as a solidifying agent, and the glucose concentration is increased to 4 g/L to support the growth of colonies. Plating dilutions that give 150-250 colonies or streaking out on MG-agar is used to isolate colonies from LTEE populations. Dilutions of the LTEE populations can also be plated on MG agar to monitor for unexpected growth, colony appearance, or CFU numbers that could indicate contamination. Ancestral clones form colonies within 24 hours on MG agar. Evolved clones typically also form colonies within 24 hours on MG agar, but some may take longer. Evolved clones also generally produce larger colonies than the ancestors on MG. MA agar: Minimal Arabinose agar is the same as MG agar except that the sugar arabinose is used instead of glucose. Ara– cells like those of strain REL606 will not form colonies on MA agar. Only Ara+ cells like those of strain REL607 will. Plating dilutions from the LTEE populations that give 150-250 CFUs on MA agar can be used to monitor the Ara– populations for contamination from Ara+ populations. The Ara+ ancestor and evolved strains generally form colonies within 24 hours on MA agar. However, some Ara+ populations have lost the ability to form colonies at later generations. Plating a large number of Ara– cells (>109) on an MA plate can also be used to select spontaneous mutants that have reverted from the Ara– marker state to the Ara+ marker state. Reversion to Ara+ among LTEE clones is usually via a single nucleotide substitution mutation in the araC gene. This mutation occurs at a rate of ~1010 cells/generation among non-mutator clones, and much higher among clones with mutator phenotypes. MC agar: Minimal Citrate agar is the same as MG/MA agar except that citrate is used as the carbon source. Strains that have evolved citrate utilization (Cit+) can form colonies on MC agar. CC agar: Christensen Citrate agar is an indicator medium that can be used to detect weak citrate utilization in colonies on the basis of a color change even, for strains that may not be able to form colonies on MC agar.