E coli top10

Sphingopyxis sp. USTB-05 used in this study was isolated and identified by us previously [45 (link)]. Trifluoroacetic acids and methanol were purchased from Dikma Technology Inc. (Lake Forest, CA, USA) and used for HPLC analysis. E. coli TOP10 and E. coli BL21 (DE3) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The recombinant and host strains were cultured in Luria–Bertani medium (tryptone 10 g, NaCl 10 g, yeast extract 5 g in 1 L H₂O) in a shaker at 200 revolutions per minute (rpm) at 30 °C. The vector pET30a (+), restriction enzymes Sac I and Not I, plasmid mini-prep kit, polymerase chain reaction (PCR) kit and Ni-NTA Sefinose (TM) Resin Kit were also obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Imidazole was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). NOD was purchased from Absin Bioscience Inc. (Shanghai, China) and stored at −20 °C. The purity of all other chemicals was no less than 99.7%.

The strain used was Sphingopyxis sp. USTB-05, capable of biodegrading MCs and NODs, previously isolated from Dianchi, China [48 (link)]. To construct a recombinant protein-expressing strain carrying the mlrB gene, we used the plasmid-amplified vector strain E. coli TOP10 and the expression strain E. coli BL21, which were purchased from Sangon Biotechnology Ltd. in Shanghai, China. Naturally, the cyclic vectors pET-30a having two histidine tags, both BamH I and Xho I restriction enzymes, the plasmid isolation kit, the polymerase chain reaction (PCR) kit, and the Ni-NTA protein purification kit were also all obtained from Sangon in Shanghai, China. The recombinant strains were grown in Luria-Bertani (LB) medium at 200 r/min at 37 °C. Ampicillin and Kanamycin were purchased from Biorigin (Beijing) Inc. (Beijing, China), which can select bacteria and prevent contamination during growth. Standard MC-LR (C₄₉H₇₄N₁₀O₁₂) of 99% purity was purchased from Shanghai Aibixin Biotechnology Co. Imidazole and standard MC-RR (C₄₉H₇₅N₁₃O₁₂, 95% purity) were purchased from Shanghai Malin Biochemistry Co., Shanghai, China. All other reagents used in the purification process were analytic grade.

CS35 and HRP-goat anti-mouse IgG antibodies were purchased from BEI Resources (USA) and Biodragon (China), respectively. ConA and BSA were obtained from Sigma–Aldrich (USA). Middlebrook 7H9 broth and oleic acid-albumin-dextrose-catalase enrichment (OADC) were purchased from BD Difco (USA). The vacuum freeze dryer was purchased from Labconco (USA). HiPrep 16/60 Sephacryl S-100 HR column was obtained from Cytiva (USA). Moreover, the 96-well plates were purchased from Greiner (Germany). 3,3',5,5'-Tetramethylbenzidine (TMB) substrate was obtained from Millipore (USA) substrate. Alexa Fluor® 594-conjugated ConA was obtained from Invitrogen (USA). All other chemicals were of analytical purity. Mycobacterium tuberculosis H37Rv (ATCC 27294), Mycolicibacterium smegmatis (ATCC 700084), and Mycobacterium bovis BCG (ATCC 35734) were preserved in our laboratory. Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 27853), and Acinetobacter Baumannii (ATCC 19606) were provided by Jianhui Li (Shanghai Public Health Clinical Center). E. coli Top10 was purchased from Sangon Biotech (China).