Infiniter m200 pro microplate reader

Putative promoter sequences were PCR amplified from M. tuberculosis H37Rv genomic DNA using Phusion High Fidelity DNA Polymerase (Finnzymes)/ KOD Hot Start DNA polymerase (Novagen) using appropriate primers (Supplementary Table S1). The PCR products were digested with XbaI and HindIII enzymes and cloned ahead of lacZ gene in promoter-less shuttle vector pCV77 in E. coli creating series of promotor fragment clones for in vivo analysis as shown in Table 1. M. smegmatis transformants containing individual clones were selected on X-gal-containing 7H11 agar plates. Independent transformants were grown in ~5 ml 7H9 broth until an OD₆₀₀ of 1. Cells were pelleted down, resuspended in 300 μl lysis buffer (1X PBS, protease inhibitor cocktail) and lysed by sonication for 3 min 15 s with 15 s pulse and 15 s rest at 30% amplitude using Sonics Vibra-Cell^TM VCX500 with 3 mm stepped microtip. The supernatant from the lysate of each transformant was analysed for β-galactosidase activity using FluoReporter^RlacZ/Galactosidase Quantitation Kit (Molecular Probes) as per given protocol. The fluorescence intensity of the samples was measured in triplicate in Nunc^TM black 96-well plates using TECAN Infinite^R M200 PRO microplate reader. The excitation and emission wavelengths used were 390 nm and 460 nm, respectively.

Selected putative terminator sequences as listed in Table 1 were PCR amplified as above (using primers listed in Supplementary Table S1), cloned in SnaBI site after GFP promoter in pVVGFPHis vector and transformed in M. smegmatis mc²155. Cloned terminator fragments include: T_tsaDF: 123 bp downstream region of tsaD- forward sequence; T_tsaDR: 275 bp downstream region of tsaD- reverse sequence; CTalr: 156 bp coding region of alr; T_synA: 40 bp synthetic hairpin sequence with a U-tract; and T_trpA: 27 bp synthetic hairpin sequence with a U-tract (based on E. coli tryptophan operon terminator). Two synthetic terminators, T_synA and T_trpA, were included to serve as positive controls for transcription termination. Three colonies of each terminator fragment were streaked onto 7H11 agar plate with kanamycin antibiotic (30 μg/ml) and then inoculated in 2 ml 7H9 broth. The cultures were grown for 3 days in a shaking incubator set at 37 °C and 200 rpm, and then sub-cultured in fresh 7H9 media with 5% inoculums. At an OD₆₀₀ of 0.8, culture volumes of 120 μl were loaded into the black 96-well microplate in triplicate. GFP fluorescence intensity was measured at 395 nm excitation and 509 nm emission wavelengths in Nunc^TM black 96-well plates using TECAN Infinite^R M200 PRO microplate reader. The fluorescence intensity values were normalized against OD₆₀₀ values.