Fig 5.

Salt-inducible production of mCherry reporter protein in recombinant H. elongata KA1-Gad and GOP-Gad strains. H. elongata OUT30018 (WT), GOP-Gad, and KA1-Gad strains cultured in M63 medium containing 4% glycerol with 3% or 6% NaCl until OD₆₀₀ reached more than 1.00 were used as a 5% inoculum for the main cultures in same salinity (3% or 6% NaCl) M63 medium containing 4% glycerol. When OD₆₀₀ of the main cultures reached more than 1.00, the cells were pelleted and subjected to tests. (A) Visualization of the salt-inducible production of the mCherry fluorescent reporter protein in the H. elongata GOP-Gad and KA1-Gad strains as shown by mCherry fluorescence of the cell pellets under fluorescent light (Fl) in comparison with bright-field (BF) images. Cell pellet of H. elongata OUT30018 (WT) was used as a negative control. (B) Detection of mCherry protein produced in the H. elongata GOP-Gad and KA1-Gad strains by western blot (WB) analysis. Proteins extracted from the cell pellets shown in (A) were electrophoresed in two identical 5%–20% gradient SDS-polyacrylamide gels. One gel was stained with Coomassie Brilliant Blue (CBB; right panels) for visualization of total protein separated on each lane, while proteins on the other gel were transferred to PVDF membrane and probed with antibody to red fluorescent protein (RFP) in WB analysis (left panels). Protein extracted from H. elongata OUT30018 (WT) was used as a negative control. Rat anti-RFP tag was used as a primary antibody, and goat anti-Rat IgG/IgM (H + L) HRP was used as a secondary antibody. mCherry protein bands were detected at 26 kDa (*).