pCC1-BAC-HIS3 plasmid BioVector NTCC质粒载体菌株细胞基因保藏中心

Name名称：BioVector® pCC1-BAC-HIS3
Description描述： pUC/pUC mini or the low-copy pCC1-BAC-HIS3 (in the case of fragment 5) were the same as described in (10). The rest of the fragments were PCR-amplified from SARSCov2 clone 3.1 ((10) except fragment 7, which was amplified from cDNA obtained by RT-PCR of viral RNA extracted from isolate USA-WA1/2020 (BEI Cat# NR-5281), grown in Vero-E6 cells. RNA was extracted from cells using Trizol (ThermoFisher #15596026) followed by RNeasy mini kit column purification (Qiagen Cat# 74104), and cDNA was prepared using SuperScript™ IV First-Strand Synthesis System with random primers (Thermo Cat# 18091050). PCR amplification reactions for fragments 1, 9-13 were performed using KOD Xtreme Hot Start DNA Polymerase (EMD Millipore Cat# 71975). Reporter/selectable marker sequences, such as mNeonGreen, Gluc, NeoR, were PCR-amplified from plasmids or purchased as synthetic DNA (IDT). Amplicons of fragments 1, 9-13 were phosphorylated with T4 polynucleotide kinase (PNK) (NEB Cat# M0201) and ligated directly into a pGEM3Z plasmid amplified via PCR (T4 DNA ligase, NEB Cat# M0202T). Fragment 7, with or without viral RdRp mutations, was cloned into the backbone of low copy number plasmid pACNR (69). SARS-CoV-2 N protein was amplified by PCR to include a polyA sequence at the 3’ end, and was cloned into KpnI/XbaI digested pGEM3Z plasmid using Gibson assembly (NEB Cat# E2611) according to the manufacturer’s protocol. Fragments-containing pUC, pUC mini and pGEM3Z plasmids were grown in E. coli DH5α (ThermoFisher), pACNR and pCC1-BAC-His3 were grown in E.coli MC1061. Plasmid DNA was extracted with ZymoPURE II Plasmid Maxiprep (Zymo research Cat# D4202). Mutagenesis of SARS-CoV-2 Nsp1 and Nsp12 was done using a two-step overlap PCR approach on fragments 2 and 7 respectively, using the primers listed in Table S1. The viral RdRp mutant was created by mutating Nsp12 catalytic residues at positions 760 and 761 from aspartic acid to asparagine (32). An Nsp1 mutant that does not bind the 40S ribosome was created by inserting K164A/H165A mutations (39, 40). The mutated fragments were used for replicon assembly as detailed below. The ΔAcc replicon was constructed using an alternative fragment 11 made via overlap PCR, which was designed to contain only E and M genes and their regulatory sequences, and none of the accessory genes. The primers for the EM-only fragment 11 are listed in Table S1. Yeast assembly and bacterial propagation of assembled replicons DNA fragments for assembly were prepared by restriction digestion or PCR as detailed in Table S1 and purified by agarose gel extraction. Yeast assembly was performed according to the protocol described in (70). Briefly, 50-100 ng of each fragment and the pCC1-BAC-His3 backbone were mixed together in an equimolar ratio and chemically transformed into Saccharomyces cerevisiae strain VL6-48N (ATCC Cat# MYA-3666). Transformed yeast cells were grown for 2-3 days on selective SD-HIS plates at 30ºC. Between 4-10 colonies from each plate were picked, re-streaked on a new selective plate and grown for 2 days at 30ºC. Crude DNA extraction was performed using glass beads (Biospec Cat# 11079110) and Chelex (SigmaAldrich Cat# 95577-100G-F) and 3 screened by multiplex PCR with the primers detailed in Table S1, using a multiplex PCR kit (Qiagen Cat# 206143) according to the manufacturer's protocol. Fragment 7, which contains the coding sequence for the viral RdRp, was amplified by PCR from positive clones and sequenceverified by sanger sequencing. For large-scale plasmid extraction from yeast, the protocol in (70) was performed as described, however ZymoPURE II Plasmid Maxiprep (Zymo research Cat# D4202) kits were used in place of Qiagen kits. To eliminate yeast chromosomal DNA, the plasmid preparation was digested with BamHI-HF enzyme (NEB Cat# R3136T), for which no restriction sites are present in the replicon-containing plasmids. Next, DNA was digested with Plasmid-Safe™ ATP-Dependent DNase (Lucigen Cat# E3101K) for 24h at 37ºC and purified via extraction with Phenol-chloroform-isoamyl alcohol (SigmaAldrich Cat# 77617), followed by ethanol precipitation. Final DNA concentration was measured using Qbit with QuDye dsDNA HS Assay (ThermoFisher Cat# Q32851) following the manufacturer’s instructions. For bacterial propagation, 1μl of the crude yeast DNA prep was electroporated into E. coli TransforMax™ Epi300™ electrocompetent cells (Lucigen Cat# EC300110). The bacteria were then plated on chloramphenicol-containing plates and grown at 30°C overnight. Bacterial clones were used to make 5ml starter cultures and grown overnight at 30°C. The following day, the starter was diluted 1:10 in fresh Luria Broth (LB) media containing chloramphenicol with copy-control solution (Lucigen Cat# CCIS125), and incubated in a shaker incubator at 37ºC for 5h. Plasmids were then extracted using the Qiaprep spin miniprep kit (Qiagen Cat# 27104). As with the yeast preps, fragment 7 was amplified by PCR and the product sequence-verified by sanger sequencing. Multiple displacement amplification (MDA) of replicon plasmids and DNA template preparation. For amplification of pCC1-BAC-His3-replicon plasmids, 5-30ng of DNA was used with EquiPhi29™ DNA Polymerase (ThermoFisher Cat# A39390) according to the manufacturer’s instructions, using exonuclease-resistant random primers (ThermoFisher Cat# SO181). Amplified DNA was digested with NotI-HF enzyme (NEB #R3189S) and purified up with phenolchloroform-isoamyl alcohol, followed by ethanol precipitation. The resulting DNA was sequenceverified by amplicon high-throughput sequencing at the Harvard MGH CCIB DNA Core Facility
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