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Subsequently, correct insertion of the mutant gene fragments into the marker free gshA mutant were checked using appropriate forward and reverse primers of the respective genes in combination with the k1 and k2 KEIO primers listed in Table 3 using colony PCR

Preparations of recombinant proteins pGEX-PGRMC1 wt, Y113F or C129S mutant expression vectors were transformed into BL21 (DE3), and the bacteria were incubated in LB with ampicillin at 37 C until OD600 reached at 0.8