E coli bl21 de3 strain

For all the molecular
cloning and precultures, Luria–Bertani broth (10 g/L tryptone,
5 g/L yeast extract, 10 g/L NaCl) was used and supplied with 100 μg/mL
ampicillin to maintain the cloned pET11a-based mutants. All cultures
were incubated at 37 °C and 300 rpm overnight.
For small-scale
production testing, 1% of mutant precultures were inoculated into
4 mL of 1x ZYM media (10 g/L tryptone, 5 g/L yeast extract, 2 mM MgSO₄, 5 g/L glycerol, 0.5 g/L glucose, 25 mM Na₂HPO₄, 25 mM KH₂PO₄, 50 mM NH₄Cl, 5 mM Na₂SO₄, and 0.02 mM FeCl₃). It was autoinduced with 15 mM lactose and supplemented with 100
μg/mL ampicillin and 34 μg/mL chloramphenicol to maintain
the mutants and p15A-cam-T7-dxs-idi, respectively.^{6 (link)} The culturing was done in 20 mL solid-phase microextraction
(SPME) vials and incubated for 20–24 h at 28 °C, 300 rpm
prior to SPME-GC/MS run.
For Ap.LS* overexpression and purification,
Ap.LS mutants were
transformed into E. coliBL21
DE3 strain carrying pTf16, a chaperone plasmid (Takara Bio
Inc., Japan). Following the transformation, 10–20 mL of overnight
preculture was inoculated into 3 L of 2x ZYM medium supplemented with
30 mM lactose and 5 mM arabinose to autoinduce Ap.LS and pTf16, respectively.
In addition, 100 μg/mL ampicillin and 34 μg/mL chloramphenicol
were added to maintain the plasmids and grown for 20–24 h at
20 °C before harvesting.