Bl21 de3

Cryptosporidium parvum oocysts (IOWA strain) were purchased from Waterborne, Inc. (New Orleans, LA, United States) and stored in antibiotics at 4°C for less than 2 months prior to use. Before experiments, oocysts were treated with 0.5% sodium hypochlorite on ice for 10 min and washed 3 times with sterile PBS. Human ileocecal adenocarcinoma HCT-8 cells were obtained from Chinese Academy of Sciences Shanghai Branch. Cells were cultured in maintenance medium at 37°C in a humidified atmosphere containing 5% CO₂. E. coli strains DH5α and BL21 (DE3) (Tiangen, Beijing, China) were used for plasmid amplification and expression, respectively. The pET28a vector was obtained from Novagen, Inc. (Madison, WI, United States). All restriction enzymes were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, United States).

The ulvan-degrading strain Alteromonas sp. A321 was provided by Peng Wang (Ocean University of China, Qingdao, China). Genomic DNA was extracted, and the whole genome was sequenced. E. coli strains DH5α and BL21 (DE3) were purchased from TIANGEN Biotech Co. (Beijing, China). The pProEX-HTa vector was used to clone and express the ulvan lyase gene. The E. coli strains were cultured in Luria-Bertani medium.

Primers used in site-directed mutagenesis were synthesized by Invitrogen Inc. (Shanghai, China). Mutations were confirmed by DNA sequencing with an ABI Genetic Analyzer 3730 (Invitrogen Inc., Shanghai, China). Escherichia coli strains DH5α and BL21 (DE3) were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). The AxyPrep Plasmid Miniprep Kit was from Axygen Biotech Ltd. (USA). The pEVOL-ONBYRS plasmid was a gift from Professor Peter. G. Schultz at Scripps Research Institute (La Jolla, CA USA). The UAAs 4-cyano-l-phenylalanine, 4-methoxy-l-phenylalanine, 4-phenyl-L-phenyalanine and O-tert-butyl-l-tyrosine were purchased from Adamas Reagent Co., Ltd. (Switzerland). Isopropyl-β-d-thiogalactopyranoside (IPTG), NADH, and acetoacetyl-CoA were purchased from Sigma Chemical Co. (St. Louis, USA).

Ampicillin, β-mercaptoethanol, imidazole, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Sangon Biotech (Shanghai, China). Tryptone and yeast extract were obtained from Oxoid (Basingstoke, Hampshire, UK). Ni-NTA agarose (catalog no. 30230) and rhodamine B (catalog no. 83689) were obtained from Qiagen (Hilden, Germany) and Sigma (St. Louis, MO, USA), respectively. Coomassie Brilliant Blue R-250 (catalog no. 161-0400) was purchased from Bio-Rad (Hercules, CA, USA). All other chemicals were of the highest purity available from commercial suppliers.
PrimeSTAR Max DNA polymerase for polymerase chain reaction (PCR) was obtained from Takara Biotechnology Co., Ltd. (Dalian, China). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA, USA). TIANprep Mini Plasmid Kit, TIANgel Midi Purification Kit, and chemically competent cells of E. coli DH5α and BL21(DE3) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Amicon Ultra-15 centrifugal filter units with Ultracel-3K membranes (3 kDa molecular weight cutoff) were obtained from Millipore (Billerica, MA, USA), and a Pierce BCA Protein Assay Kit (catalog no. 23225) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA).

His or GST fusion proteins were expressed in BL-21(DE3) (Tiangen Biotech) according to the manufacturer’s instructions. GST fusion proteins were lysed by ultrasonication, incubated with GST beads for 4 h at 4 °C, and then washed three times with TBST to collect the precipitates. Purified His fusion protein or lysates of mouse peritoneal macrophages were incubated with the collected precipitates for 4 h at 4 °C. After centrifugation and washing, the beads were boiled in 1× SDS loading buffer at 100 °C for 10 min and then analyzed by immunoblotting.

His or GST fusion proteins were expressed in BL-21(DE3) (CB105-02; Tiangen Biotech) according to the manufacturer’s instructions. His fusion protein was purified with Ni Sepharose affinity chromatography. GST fusion proteins were lysed by ultrasonication, incubated with GST beads for 4 h at 4°C, and then washed three times with TBST to collect the precipitates. Then, purified His fusion protein incubated with the collected precipitates for 12 h at 4°C, centrifugation and washing, the beads were boiled in 1× SDS loading buffer at 100°C for 10 min and then analyzed by immunoblotting.

Shiga toxin-producing E. coli (STEC) F107/86 (O139:H1) [26 (link)], Enterohemorrhagic E. coli (EHEC) EDL933 (O157:H7) [27 (link)], and enterotoxigenic E. coli (ETEC) C83902 (O8:K88:H19) [28 ] were used to amplify the fliC_H1, fliC_H7, and fliC_H19 gene, respectively. They were grown in Luria–Bertani (LB) broth or on LB agar plates at 37 °C. Expression vector pET28α ( +) (Novagen, USA) and E. coli strains DH5α and BL21 (DE3) (TIANGEN, China) were used for gene cloning and subsequent recombinant protein expression.