Plasmid extraction kit

E. Coli bacteria with clathrin-LCa-EYFP plasmid was acquired from Addgene (Plasmid 21741, Cambridge, MA)^{41 (link)}. The plasmid was extracted from the bacteria by using plasmid extraction kit (cat. 27104) and the corresponding protocol from QIAGEN (Valencia, CA). The A549 cells were pre-grown on coverslip in a Petri dish for 24 h in the incubator. The EYFP-clathrin plasmid was mixed with the transfection reagent (FuGene6, Roche Applied Science, Indianapolis, IN) in serum-free cell culture media first and introduced into the Petri dish containing the cells. After additional 24–48 h incubation, the cells were ready to use. In control experiments, the cells were incubated with hypertonic media (0.45 M Sucrose) or clathrin coat disruption drug (10 µg ml⁻¹ Chlorpromazine Chloride) at 37 °C before exposure to the surface-modified gold nanorods.

E. coli BL21(DE3) was obtained from MerckMillipore (Burlington, MA, USA). pET28a expression vector was purchased from Invitrogen (Waltham, MA, USA). Fetal bovine serum (FBS) and RPMI 1640 were purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). Plasmid extraction kit and Ni-NTA-agarose column were obtained from Qiagen (Hilden, Germany). The luciferase assay kit was purchased from Promega (Madison, WI, USA).

Plasmids of the bla_CTX–M–55-positive transconjugants were extracted using the Plasmid Extraction Kit (Qiagen, Hilden, Germany). The plasmids recovered from transconjugants were sequenced by the Illumina NextSeq 500 platform and the MinION sequencing platform. The method used to perform MinION sequencing was reported previously; Unicycler was used to perform hybrid assembly (Wick et al., 2017 (link); Li et al., 2018 (link)). The completed plasmid sequence was confirmed by PCR and then annotated with the RAST tool and the NCBI Prokaryotic Genome Annotation Pipeline. BLAST tools were used for the comparative analysis of plasmid sequence and ARI-A sequences.

HEK293 cells and HeLa cells were purchased from ATCC, cultured in DMEM containing 10% fetal bovine serum and 1% Penicillin-Streptomycin. All culture reagents were purchased from GIBCO (USA). The inserted gene was synthesized by Synbio Technologies Co., Ltd. (China). pAAV-MCS, pAAV-RC9, and pAAV-Help were purchased from Biofeng (China). The transfection reagent Lipofectamine 3000 was purchased from Thermo Fisher (USA). Three plasmid transfection reagents, FectoVIR-AAV, were purchased from Polyplus (USA). The anti-RBD monoclonal antibody was obtained from Sino Biological (China). The plasmid extraction kit was purchased from QIAGEN (USA). The AAVpro Titration Kit (for Real Time PCR) Ver.2 was obtained from TaKaRa (Japan). The benzonase and iodixanol reagents were purchased from Sigma (USA). The luciferase detection reagent was obtained from PerkinElmer (USA). The novel corona pseudovirus and Huh-7 cells were donated by Professor Wang Youchun and Professor Huang Weijin, respectively, from the National Institutes for Food and Drug Control.

BAC clones containing the target genes were selected from the UCSC Genome Bioinformatics Library and confirmed by PCR. Briefly, the BAC DNAs were prepared using a Qiagen plasmid extraction kit and digested with EcoRI before labeling with fluorochrome-linked dUTP (Table 4) by nick translation.