Ecor1

A 822 bp of cDNA fragment consisting of the cap gene that encodes the full-length PiCV capsid protein was synthesized by Genemark Biosci & Tech Co. (Taichung, Taiwan) based on the published sequence (Columbid circovirus, isolate 9030; Accession No. AJ298229). This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using EcoR1 and XhoI (Takara, Japan) restriction sites. The resulting recombinant plasmids were designated pHis-Cap, pTrx-His-Cap and pGST-Cap, respectively (Figure 1, panel a, e and c). To improve the codon usage of the cap gene from PiCV, a second cDNA sequence was synthesized by Genemark Biosci & Tech Co that contained the codons that were optimized for E. coli; this was also ligated individually into the same three E. coli expression vectors using the same restriction sites; these constructs were designated pHis-Cap_opt, pTrx-His-Cap_opt and pGST-Cap_opt, respectively (Figure 1, panel b, f and d). The six constructs were then individually transformed into One Shot^® Top10 (Invitrogen, CA) chemically competent E. coli for maintenance of the recombinant plasmids. Transformants that containing a insert of the correct size were then confirmed as correct by restriction enzyme digestion and by DNA sequence analysis.

Carthamus tinctorius cultivar, Jihong No.1 seeds were purchased from Fuyu Seeds Company, China. Wild-type A. thaliana was grown for 5–7 weeks, and the early flowering plants were subjected to floral-dip transformation. Agrobacterium tumefacien strain EHA105, Escherichia coli BL21, E. coli DH5α, the prokaryotic expression vector (PET28a-CtCYP82G24), (the plant over-expression vector pBASTA-CtCYP82G24), the subcellular localization vector (CtCYP82G24-pCAMBIA1302-GFP (green fluorescent protein) was successfully constructed and preserved the 80% glycerol stock at −80 °C until next use. Restriction enzymes (BamH1, EcoR1, BglII, and Spe1) and DNA ligase (T4) were purchased from Takara Biotechnology Company Beijing, China.

The pNIM-001 plasmid was linearized by EcoR1 (Takara) which does not target any sequence in the PCR amplicon before analyzing its quantity using four digital PCR platforms. Enzymatic digestion of mixture comprised of 2 μL 10× buffer, 1 μL EcoR1 (15 U/μL) restriction enzyme, 10 μL plasmid pNIM-001 DNA, and 7 μL ddH₂O. No template control (NTC) was prepared by adding 10 μL 1 × TE_0.1 (10 mM Tris-HCl, 0.1 mM EDTA, pH = 8.0) instead of the DNA solution, and no enzyme control (NEC) was made by pipetting 1 μL 1 × TE_0.1 in place of the enzyme when preparing the enzymatic master mix. The enzymatic reaction lasted for 1 h at 37 °C and inactivated for 15 min at 65 °C. After the enzymatic reaction, the DNA was diluted to suitable concentrations for analysis on the different digital PCR platforms. Same plasmid DNA solutions with different concentration were mixed with prime probe and PCR master mix to be analyzed on four digital PCR platforms.

Restriction enzymes (EcoR1 and Xba1), T4 DNA ligase, DNA marker were purchased from Takara (Dalian, China). The protein marker was purchased from Thermo Fermentas (Guangzhou, China). E. coli DH5α, Pichia pastoris GS115 pPICZαA vector were stored by our laboratory. Zeocin antibiotic was obtained from Invitrogen (Carlsbad, CA). Anti-Bcl2, anti-Bax, anti-GAPDH, anti-Myc, HRP-goat anti-rabbit conjugate were purchased from Cell Signaling Technology (Danvers, MA). HEK-293 human primary embryonic kidney cells, human breast cancer cell MDA-MB-231 and MCF-7, human liver cell LO2, human liver cancer cell SW460, human glioma cell SHG-44 and human cholangiocellular carcinoma cell Hucc-T1 were obtained from the American Type Culture Collection. All the other chemicals were of analytical grade. Annexin V-FITC apoptosis detection kit was a product of BD Biosciences, USA.

The pBR322, pNIM-001, and pNIM-002 plasmids were linearized by EcoR1 (Takara) that does not target any sequence in the PCR amplicons. Enzymatic digestion mixture was comprised of 2 μL of 10 × buffer, 1 μL of EcoR1 (15 U/μL) restriction enzyme, 10 μL of plasmid DNA, and 7 μL of ddH₂O. Non-DNA and non-enzyme controls were prepared by replacing the DNA and enzyme with 10 μL of 1 × TE_0.1 (10 mM Tris-HCl, 0.1 mM EDTA, pH = 8.0) and 1 μL of 1 × TE_0.1 in the master mix, respectively. The enzymatic reaction was carried out for 1 h at 37 °C, and inactivated for 15 min at 65 °C. After the enzymatic reaction, the DNA was analyzed on the qPCR or dPCR instrument. Agarose gel analysis with fluorescent staining was used to check if the restriction digestion was completed, the electrophoresis image was in Fig. S1 (in supplemental material).