Measuring PAL Activity and Phenylpyruvate

Example 64

A 1:100 back-dilution from overnight culture of SYN-PKU-2002 was grown to early log phase for 1.5 h before moving to the anaerobic chamber for 4 hours in the presence of 1 mM IPTG and 0.1% arabinose for induction as described herein. To perform activity assay, 1e8 cells were resuspended and incubated in assay buffer (M9 media with 0.5% glucose, 50 mM Phe, and 50 mM MOPS with 50 mM phenylalanine). Supernatant samples were taken over time and TCA (the product of PAL) was measured by absorbance at 290 nm to determine the rate of TCA production/PAL activity. Phenylpyruvate was measured using LCMS methods described herein. Results are shown in FIG. 16A and FIG. 16B.