Expanding the bioluminescent reporter toolkit for plant science with NanoLUC

Recombinant expression of NanoLUC variants and characterisation. a 1.5 ml microcentrifuge tubes containing E.coli Rosetta 2 BL21 pLyS pET28a:NanoLUC cells induced with IPTG (+) or without IPTG (-) after being incubated for 6 h at 30 °C 200 r.p.m. Photo taken with a standard mobile phone camera to exemplify NanoLUC brightness. b Purification of NanoLUC-3× FLAG-10× His (NL3F10H, ~？23.53 kDa) by IMACS, wash steps done with wash buffer, elution 1 and 2, performed with 200 μl of elution buffer. c Purification of Maltose-Binding-Protein-NanoLUC-3× FLAG-10× His (MBP-NL3F10H, ~？66.83 kDa) by IMACS. Purification described in methods. 10 μl of each purification fraction was loaded into a NuPAGE 4-12% Bis–Tris (Invitrogen) and run at 200 V for 40 min. d Linearity of NanoLUC invitro assay. Protein was quantified by linearized Bradford assay and adjusted to 10 mM. Serial dilutions were generated for purified NL3F10H or MBP-NL3F10H shown in B and C, enzymatic assays were performed as in F. e Effect of Col-0 plant extract on the activity of NanoLUC. Black circles, serial dilutions of purified MBP-NL3F10H in plant extract of 21 days old Col-0 plants. The extract was obtained by liquid nitrogen freezing and grinding in Tissue lyser. Grinded tissue was resuspended in BSII buffer to 0.4 gFW ml？1. Blue circle, 1:1000 dilution of MBP-NL3F10H in BSII buffer. f Stability of NanoLUC. MBP-NL3F10H was stored at 4 °C in Elution buffer (250 mM NaCl, 50 mM NaH2PO4 pH 8.0 NaOH adjusted, EB). The purified enzyme was diluted 1:1000 in Elution buffer and mixed with 100 μl of 1:50 furimazine:NanoGlow buffer in a 96-well flat black plate (Lumitrac, Greiner). Samples were incubated for 10 min at 21 °C in a Tristar luminometer plate reader. Solid blue line fitted exponential decay model results in a half-life of ~？37 days. Circles, mean of three biological replicates, error bars S.E.