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.