E coli dh5α competent cells

DNA fragments that encode SARS-CoV-2 variant RBD (Spike 319–541) were codon-optimized for human cell expression and synthesized by Genscript. His-AVI tags were added at the end of the fragments. The fragments were then inserted into pCMV3 vector through infusion (Vazyme). The recombination products were transformed into E. coli DH5α competent cells (Tsingke). Colonies with the desired plasmids were confirmed by Sanger sequencing (Azenta) and cultured for plasmid extraction (CWBIO). 293F cells were transfected with the constructed plasmids and cultured for 6 days. Products were purified using Ni-NTA columns (Changzhou Smart-lifesciences, SA005100) and the purified samples were verified by SDS-PAGE.

Twelve Bacillus strains preserved in the laboratory, including 3 strains of Bacillus subtilis, 2 strains of Bacillus atrophaeus, 2 strains of Bacillus sonorensis, 3 strains of Bacillus amyloliquefaciens, Bacillus aryabhattai, and Bacillus methylotrophicus, were used for gene screening. The pMD-19 T vector (Takara, Dalian, China) was used for cloning and sequencing. E. coli DH5α competent cells (Tsingke, Beijing, China), E. coli BL21(DE3) cells (Vazyme, Nanjing, China), the pET28a(+) vector (Yuanye, Shanghai, China), and the pET-duet vector were used for protein overexpression. Restriction enzymes Nde I, Not I, Mlu I, Sac I, Sal I, BamH I, and EcoR I (Takara) and T4 DNA ligase (Vazyme) were used for plasmid manipulation and the ligation reaction, respectively. Substrates and standard chemicals were purchased from commercial sources, including 2,2’-azinobis(3-ehtylbenzothiazolin-6-sulfnic acid) ammonium salt (ABTS) (Aladdin, China); naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, and benzo[a]pyrene (Macklin, China); and ampicillin, kanamycin, and isopropyl-beta-D-thiogalactopyranoside (IPTG) (Solarbio, Beijing, China).