2.2. Plasmid construction, strains, media, and culture conditions
E. coli DH5α was used as the host for genetic engineering. Cells were cultured and maintained at 37 °C in lysogeny broth (LB) containing 10 g/L tryptone, 5 g/L yeast extract, and 5 g/L sodium chloride. Antibiotics were added as required (50 μg/mL kanamycin, 100 μg/mL spectinomycin, 35 μg/mL chloramphenicol, and 50 μg/mL ampicillin).
pipA encoding LCD was amplified from Streptomyces pristinaespiralis ATCC25486 chromosomal DNA as the PCR template. PCR products were inserted into the MCS-1 site of pCDF duet-1 vector via digestion with BamHI and SacI, and into the MCS-2 site via digestion with MfeI and KpnI. The constructed plasmids were prepared and transformed into E. coli BL21 Star (DE3) competent cells for the whole-cell reaction. Engineered plasmids were confirmed with sequencing (Cosmo Genetech, Seoul, Korea). Bacterial strain and plasmid information is listed in Table 2.
E. coli BL21 Star (DE3) bacteria harboring the constructed plasmid were cultured at 37 °C in a shaking incubator (Han‐Beak Science Co., Bucheon, Gyeonggi‐do, Korea) at 200 rpm. The culture was incubated overnight in 5 mL LB medium with appropriate antibiotics for plasmid stability in a 14 mL round‐bottom tube. The incubated cells were added to 50 mL terrific broth (TB) with antibiotics in a 250 mL baffled Erlenmeyer flask and incubated at 37 °C with shaking. At an OD600 of 0.6–0.7, protein expression was induced with 0.1 mM isopropyl β‐D-1‐thiogalactopyranoside (IPTG), and the temperature was decreased to 30 °C. After 16 h, the cultured cells were harvested, centrifuged at 5,500 rpm for 10 min at 4 °C, washed, and resuspended with deionized water to an OD600 of 100.