Figure 2.

Optimizing conditions of growth-based PAL selection. (a.) E. coli MG1655(DE3)^{ΔendA,ΔrecA} cells expressing AvPAL* (blue) or truncated AvPAL* (orange) were grown in MM^phe under different conditions. Growth rates (OD₆₀₀/day) are in the top right corner with optimum conditions shaded. (b.) The final optimized conditions allowed growth recovery in 12 h compared to 3 d previously. (c.) While optimizing the growth media, we observed that at phe > 30 mM, final biomass density decreased and lag time increased, suggesting toxicity due to rapid accumulation of tCA. At phe < 30 mM, final biomass densities dropped, and when ≤ 7.5 mM, growth rate also slowed, suggesting insufficient nitrogen to sustain growth. (d.) Media supplemented with tCA inhibited the growth of E. coli at concentrations ≥ 1 mM. (e.) AvPAL* expressing cells produce and secrete ~ 1 mM tCA before arresting growth. The subsequent bolus increase in tCA during death phase is likely due to cell lysis. All curves representative of duplicates with less than 10% error.