Chloramphenicol → isopropyl beta-D-thiogalactopyranoside

Participants

Reaction Steps

All liquid cultivations were conducted in MOPS minimal medium (Teknova) [14] cultures supplemented with 5 g/l glucose and an additional 4 g/l NH4Cl. When appropriate, antibiotics were added in the following concentrations: 100 μg/ml carbenicillin for the maintenance of pTrc- or pET-derived plasmids, 34 μg/ml chloramphenicol for pHACM-derived plasmids, 68 μg/ml chloramphenicol for pACYC-derived plasmids and pRARE2 (Novagen), and 20 μg/ml kanamycin for pACKm-derived plasmids. Isopropyl-β-D-thiogalactopyranoside (IPTG, EMD Chemicals) was provided at a concentration of 1 mM for the induction of expression from both trc and T7 promoters. Cultures for L-phenylalanine auxotrophic (ΔpheA) strains were additionally supplemented with L-phenylalanine (Sigma) at a concentration of 0.35 mM. For malonyl CoA availability experiments, cerulenin (Cayman Chemicals) and sodium malonate dibasic (Sigma) were added at a concentration 20 μg/ml and 2 g/l (1 g/l added twice), respectively.

Provenance & Evidence