Ni nta chromatography

The sgnE gene was amplified from S. gilvosporeus F607 genomic DNA using the primer pair sgnE His-F/R (Table S2) and then cloned into pMD18T to generate pMD18T-sgnE. After confirmation by DNA sequencing, sgnE from pMD18T-sgnE was cloned into the expression vector pET-15b to construct pET-sgnE, which was transformed into E. coli BL21 (DE3) to obtain E. coli BL21/pET-sgnE. When E. coli BL21/pET-sgnE was cultured to an OD_{600 nm} value of 0.6–0.8, 0.1 mM isopropyl-d-1-thiogalactopyranoside was added to induce SgnE expression. His₆–SgnE was purified by Ni-NTA chromatography (Sangon Biotech, Shanghai, China) (46 (link)). Construction of recombinant E. coli BL21/pET-OxyR and expression and purification of OxyR were performed using analogous procedures. Two fragments (amplified using the primers C212D-F and C212D-R) and pMD18T were linked using Gibson Assembly Master Mix and assembled with pET-15b to obtain the Cys212Asp variant of OxyR, following the same procedures.

The sgnE gene was amplified from S. gilvosporeus F607 genomic DNA using the primer pair sgnE His-F/R (Table S2) and then cloned into pMD18T to generate pMD18T-sgnE. After confirmation by DNA sequencing, sgnE from pMD18T-sgnE was cloned into the expression vector pET-15b to construct pET-sgnE, which was transformed into E. coli BL21 (DE3) to obtain E. coli BL21/pET-sgnE. When E. coli BL21/pET-sgnE was cultured to an OD_{600 nm} value of 0.6–0.8, 0.1 mM isopropyl-d-1-thiogalactopyranoside was added to induce SgnE expression. His₆–SgnE was purified by Ni-NTA chromatography (Sangon Biotech, Shanghai, China) (46 (link)). Construction of recombinant E. coli BL21/pET-OxyR and expression and purification of OxyR were performed using analogous procedures. Two fragments (amplified using the primers C212D-F and C212D-R) and pMD18T were linked using Gibson Assembly Master Mix and assembled with pET-15b to obtain the Cys212Asp variant of OxyR, following the same procedures.