Top10 chemically competent e coli cells

DNA plasmids (pVAX1, pGag, pA27L, and pOD1A27Lopt) were propagated by transformation of TOP10 Chemically Competent E. coli cells (Invitrogen, Valencia, CA, USA). PureLink® HiPure Plasmid DNA GigaPrep Kit (Invitrogen, Carlsbad, CA, USA) was used for plasmid purification. The plasmids were resuspended in ultrapurified water and stored at -20°C until further use.
The integrity of constructs was verified by double enzymatic digestion (New England Biolabs, Ipswich, MA, USA) and DNA sequencing (Molecular Biology Core Facility, UPR: RCM-RCMI Program, San Juan, PR, USA). The agarose gel electrophoresis separation showed the expected 1.6 kbp (pGag), 0.4 kbp (pA27L), and 1.4 kbp (pOD1A27Lopt) bands (Figure S2). Assembly validation was completed using the Bioinformatics Software MacVector (MacVector Inc., Apex, NC, USA).

The purified PCR products were ligated into the pCR2.1-TOPO vector and transformed into TOP10 chemically competent E. coli cells according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). The transformed cells were plated on Luria-Bertani (LB) agar plates containing 100 μg mL^-1 of ampicillin, 80 μg mL^-1 of 5-bromo-4-cholo-3-ondolyl-b-D-galactopyranoside (X-Gal). After an overnight incubation at 37°C, white transformants were transferred to LB ampicillin (100 μg mL^-1) agar plates and cultured overnight. To ensure the presence of the insert, PCR amplification was performed directly on the colonies by using specific primer pair for each target. The plasmids from a random selection of positive clones were purified and sequenced using M13 universal primers (Invitrogen) and the Taq Dye-Deoxy Terminator Cycle Sequencing kit (Life Technologies Co., Carlsbad, California, United States). The forward and reverse sequences for each target were obtained loading the respective samples on an ABI 310 Genetic Analyzer (Life Technologies Co., United States).

Viral RNA was extracted from 140 μL of cell supernatant using a QIAamp^® Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and stored at −80 °C until it was used. Quantitative analysis of purified RNA was performed using the in-house real-time RT-PCR protocol described by Skittrall [70 (link)], targeting a 222-base region within the RNA-dependent RNA polymerase (RdRp) region of the SARS-CoV-2 1ab gene. A standard curve was generated using duplicate serial dilutions (10⁸–10¹) of the SARS-CoV-2 NSP12 VersaClone cDNA plasmid (R&D Systems, Minneapolis, MN, USA; catalog no. RDC3140), propagated in TOP10 Chemically Competent E. coli cells (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and quantified in an Eppendorf BioPhotometer (Hamburg, Germany) (ηg/μL) after purification using a QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer’s instructions. Reactions were run on an Applied Biosystems 7500 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA).

Total RNA was isolated from blood stages/host white blood cell mixtures with RNeasy Mini Kit (Qiagen, Czech Republic), for transcriptome sequencing at Beijing Genomics Institute (BGI, Hong Kong) with Illumina HiSeq platform. The Trinity assembly will be published in another publication however the Fragments Per Kb per Million fragments (FPKM) was performed to screen for transcripts with high abundance. Actin transcripts were confirmed in a separate cDNA sample produced by the SMART cDNA Library Construction Kit (Clontech, Czech Republic), by sequencing PCR amplicons produced with degenerate actin primers ACT-F (AAC TGG GAY GAY ATG GAR AAG AT) and ACT-R (ATC CAC ATY TGY TGG AAN GT)59 (link). The same primers were used to amplify beta actin sequences from DNA extractions of myxozoan spores belonging to other species (Table 1). PCR conditions were: 5 mins at 94 °C, 30 cycles of 94 °C 1 min, 53 °C for 1 min, and 72 °C for 1 min, with a final extension of 5 mins at 72 °C. Amplicons were cloned into the pDrive Vector (Qiagen, Germany), transformed with TOP10 chemically competent E. coli cells (Life Technologies, Czech Republic), and 15 clones per species were sequenced commercially in both directions with M13 primers (https://www.seqme.eu).

A gBlock Gene Fragment (Integrated DNA Technologies, Coralville, IO, USA) was designed containing the amplicons for the four phage species, Erwinia amylovora, and Pantoea agglomerans listed in Table 4. The ends each contain an EcoRI restriction site with an additional external 10 random bases to aid EcoRI digestion. The fragment was cloned into a pIDTBlue vector modified with an inserted EcoRI site to create the plasmid pTotalStdA (Figure 1). The construct was transformed into TOP10 Chemically Competent E. coli cells (Life Technologies, Carlsbad, CA, USA). The transformed cells were grown in 2% LB broth (BD, Sparks, MD, USA) with 200 µg/mL ampicillin overnight at 37 °C, 200 rpm. Plasmid isolation was carried out using the Qiagen Plasmid Mini Kit (Qiagen, Toronto, ON, Canada) as per manufacturer’s instructions. The plasmid was linearized using ScaI restriction enzyme (New England Biolabs, Ipswich, MA, USA), quantified using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and diluted to 10¹² copies/mL in TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA). A log₁₀ dilution series was prepared in TE and analyzed with qPCR for all six primer and probe sets to ensure proper dilution and amplification.

pArs_lac was created using pArs as a template by using primers containing a pArs-specific sequence and part of the lac operator sequence. The primers used (“Lac operon forward” and “Lac operon reverse,” Figure S3 in Supplementary Material) were designed to be back-to-back, thereby producing full-length linear pArs plasmid with the lac operator sequence overhanging, half at the 3′ and half at the 5′ end. PCR conditions: initial denaturation 98°C, 30 s; 35 cycles of 98°C, 30 s, 72°C, 3 min 30 s; final elongation 72°C, 10 min. The primers were phosphorylated at the 5′ end so that after PCR, the linear vector could be circularized using T4 DNA ligase, and then transformed into E. coli TOP10 chemically competent cells (ThermoFisher), and subsequently transferred to E. coli JM109 cells.