Nico21 de3 competent escherichia coli

The cDNA of either oFluc (RDB14359 from RIKEN BRC) or Venus/Akaluc^{26 (link)} was inserted in the multiple cloning site of pRSET-B using BamHI and EcoRI (Thermo Fisher Scientific). The luciferase protein expressed in NiCo21(DE3) competent Escherichia coli (New England Biolabs) was purified using a Ni-NTA agarose resin column (Qiagen) and a chitin resin column (New England Biolabs). Protein concentrations were measured using a Bradford Protein Assay (Bio-Rad). Emission spectra were determined using a LumiFl-Spectrocapture AB-1850 instrument (Atto) at 37 °C in a solution of 0.1 M citrate phosphate buffer (pH 7.0) containing the substrate (1 mM d-luciferin or 10 µM AkaLumine), 1 mM ATP, 2 mM MgSO₄ and each of the purified luciferases (10 ng/µl in 100 µl reaction volume).

The pET47b(+)-MS2-ZDC vector was transformed into NiCo21(DE3)-competent Escherichia coli in accordance with the manufacturer's instructions (New England Biolabs, USA). The protocol for expression and purification was described previously by Zambenedetti et al. [12 (link)], with some modifications. The expression of pET47b(+)-MS2-ZDC was induced by the addition of 0.5 mM isopropyl-1-β-D-thiogalactoside (IPTG), and after centrifugation, the supernatant was collected and processed with filtration for viral-like particles purification. After purification, viral-like particles were stored at –20°C.