Isopropyl β d 1 thiogalactopyranoside (iptg)

Plasmid pHGEN-Ptac was used for genetic complementation of the mutants generated in this study⁴² . Genes of interest were generated by PCR, cloned into the vector, and the resultant vectors were transformed into E. coli WM3064. After verification by sequencing, the vectors were transferred into the relevant S. oneidensis strains via conjugation. Expression of the cloned genes was controlled by IPTG (Abcam Shanghai)-inducible promoter Ptac. For expressing S. aureus aa₃, the qoxABCD operon was first cloned behind Ptac and then the ctaM gene was placed after the operon but before the terminator sequence.

NaHS was obtained from Sigma Chemical (St. Louis, MO, USA). Tris-Base was purchased from Amersco (St. Louis, MO, USA). Ethanol, glacial acetic acid, Na₂S₂O₃, sodium acetate, glutaraldehyde, silver nitrate, formaldehyde, sodium carbonate, and dithiothreitol (DTT) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cell counting kit-8, ATP, PLK1, PKA, and IPTG were purchased from Abcam (San Francisco, CA, USA). The LDH assay kit was purchased from Servicebio (Wuhan, China). The NSE assay kit was obtained from Jiangsu Meimian Industrial, Co., Ltd. (Jiangsu, China). Flou-8 AM was purchased from biofount (Beijing, China).

Controlled gene expression was used in genetic complementation of mutants and assessment of physiological effects of proteins at varying levels. Genes of interest were generated by PCR, cloned into plasmid pHGEN-Ptac under the control of Isopropyl β-D-1-thiogalactoside (IPTG)-inducible promoter Ptac, and the resultant vectors were transformed into E. coli WM3064 (Meng et al., 2018a (link)). After verification by sequencing, the vectors were transferred into the relevant S. oneidensis strains via conjugation. Expression of the cloned genes was controlled by IPTG (Abcam, Shanghai) at varying concentrations.