Genios pro multifunction microplate reader

Rat cortical astrocytes were transfected at 13 DIV using 190 ng of luciferase reporter plasmid (or 380 ng of DNA when cotransfected with effector plasmids) and 10 ng of pGL4.83‐SRα‐hRLuc normalizer plasmid (or 20 ng when cotransfected with effector plasmids) in unsupplemented DMEM with a DNA to Lipofectamine 2000 (Invitrogen) ratio of 1:3 (or 1:2 for cotransfection of reporter plasmid and effector plasmid). The transfection was terminated by changing the media to fresh supplemented DMEM.
After treatments at 15 DIV the cortical astrocytes were lysed in 1× Passive Lysis Buffer (Promega) and luciferase assay was performed using Dual‐Glo® Luciferase Assay (Promega) system. Luminescence signal was measured using GENios Pro Multifunction Microplate Reader (Tecan). For data analysis, background corrected Firefly luciferase signals were normalized with background corrected Renilla luciferase signals and the averages of duplicates were calculated.

To construct fhA1, tssA1 and pelA’-‘lux translational fusions, ∼0.5-kb fragments generated from PAO1-N chromosomal DNA by PCR with primers MiniCTXluxFha1Fw/Rv, MiniCTXluxTssa1Fw/Rv and MiniCTXluxPelAFw/Rv were cloned into XcmI-cut miniCTX-lux(Gm^r) plasmid by in vitro homologous recombination using a Gibson assembly kit (New England BioLabs). Plasmids obtained were mobilized from E. coli S17-1 λpir and mini-CTX elements inserted in the chromosome of PAO1-N Wild-type (WT) and the ΔrsmN, ΔrsmA and ΔrsmArsmN_ind mutants by mating. To semi-quantify c-di-GMP levels the plasmid PcdrA::gfp^S was mobilized into PAO1-N Wild-type (WT) and the ΔrsmN, ΔrsmA and ΔrsmArsmN_ind mutants respectively. Clones with active fusions were selected and analyzed for bioluminescence or fluorescence output activity over growth in LB at 37°C using a 96-well plate TECAN Genios Pro multifunction microplate reader.