Plasmid mini kit 1

The primers used for standard recombinant plasmid construction are listed in Table 1. Viral DNA fragments were recovered from agarose gels after the polymerase chain reaction (PCR) had completed. The recovered, purified products were ligated into pMD19-T, then the plasmid pMD19-T-VP1 was extracted using a Plasmid Mini Kit I (OMEGA Bio-Tek, Norcross, GA, USA).

Endo H_f and PNGase F were purchased from New England BioLabs. p-nitrophenyl palmitate (pNPP), endoproteinase Asp-N and trypsion were obtained from Sigma (USA). Horseradish peroxidase-conjugated goat anti-mouse IgG, Anti-His antibody and Pro-LightHRP chemical reflective detection reagents was purchased from TianGen Biotech (Beijing, China). Western blotting Marker and nitrocellulose membrane (PVDF) were obtained from BIO-RAD. Dpn I, PrimeSTAR polymerase, PCR reagents were obtained from Takara Biotechnology (Dalian, China). SDS-PAGE Protein Marker was provided by Beyotime Institute Biotechnology. Primers were synthesized at Sangon Bitech (Shanghai, China). Gel extraction and PCR purification kits were purchased from Bioflux (Hangzhou, China). A Plasmid Mini Kit I was obtained from OMEGA Bio-Tek. A One Step Yeast Active Protein Extraction Kit was purchased from Sangon Bitech. All other chemicals used were of the highest quality that is commercially available.

A partial ssrA fragment of Bartonella was cloned into the pEASY-T1 vector (TransGen Biotech, Beijing, China), and the resulting T-loaded construct was transformed into DH5α E. coli cells. Following bacterial liquid amplification, the presence of the inserted target gene was confirmed through sequencing analysis. A small quantity of plasmids carrying the cloned ssrA fragment were extracted using the Plasmid Mini Kit I (Omega Bio-tek, Norcross, GA, USA) and stored at −80 °C for future use. Upon melting, the concentration of Bartonella plasmids extracted was determined by an ultraviolet spectrophotometer (Life Real, Hangzhou, China). The concentration was then converted into a copy number using the formula:
Copies/µL = Plasmid concentration (ng/µL) × 10⁻⁹ × 6.02 × 10²³/(660 × DNA length)
The plasmid concentration was converted into a copy number, which was used for the establishment of a standard curve and quantitative analysis as the positive control.

The ITS region of A. bombi (originating from an infected Tetragonisca fiebrigi colony of Rolante) was cloned using the CloneJET PCR Cloning Kit (Life technologies, Gent, Belgium) and isolated with the Plasmid Mini Kit I (Omega Bio-Tek, Norcross, GA, USA). Sequence was obtained by sequencing of plasmids with pJET1.2 primer and were BLAST-searched for confirmation. Sequence was aligned with other ITS sequences from NCBI using ClustalW and subsequently trimmed in MEGA633 (link). Phylogenetic tree was constructed with maximum likelihood using the Hasegawa-Kishino-Yano model.

The Widom 601 sequence^{44 (link)} was inserted into pET-51b vector, and the resulting plasmid was used as PCR template. The plasmid was transformed into Trans1-T1 E. coli cells and isolated using Plasmid Mini Kit I (Omega Bio-Tek). Damaged base-containing Widom 601 DNA was prepared by PCR with DI-containing primers. The primers used in this study were listed in Supplementary Table S2. A typical purification procedure required 10 mL of PCR reaction product. PCR reaction product was loaded onto a 5-mL Q beads 6FF FPLC column (Smart-Lifesciences, Changzhou) pre-equilibrated with 20 mM Tris-HCl (pH 8.0), and then eluted with linear gradient of NaCl (from 0 to 2 M, 20 mM Tris-HCl, pH 8.0). Peak fractions containing Widom 601 DNA were collected and concentrated using 10 K AmiconUltra-15 centrifugal filter unit (Merck).
Concentrated solution was supplemented with 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol to precipitate DNA. After 1 h of freezing at –40 °C, the mixture was centrifuged at 15000 rpm at 4 °C for 10 min, and the DNA pellet was washed with ice-cold 70% ethanol and dried in air. The resulting DNA pellet was stored at –80 °C for nucleosome reconstitution.

According to the method we previously described [13 (link)], it was amplified that the 47 kDa gene of 1401 bp of the O. tsutsugamushi Karp strain, and were cloned into the pEASY-T1 vector (TransGen Biotech, Beijing, China), and the T-loaded products were transformed into DH5α E. coli cells. E. coli cells containing target DNA on LB medium were grown in LB medium for 9 hours. Plasmid DNA was extracted from 600μl of the suspension, using the Plasmid Mini Kit I (Omega Bio-Tek, Norcross, GA, America), following the manufacturer’s instructions and the concentration was determined. The O. tsutsugamushi data in ng/μl were then converted to numbers of copies/μl. For plasmid detection sensitivity, we performed real-time quantitative polymerase chain reaction (qPCR) with serial dilutions of plasmid DNA from 5×10⁹ copies/μl to 5×10³ copies/μl using DNase-free deionized water to create a standard curve.
The presence of O. tsutsugamushi was assessed by qPCR and the degree of infection in the host animals was determined using the 47 kDa high temperature transmembrane protein gene. qPCR was accomplished using probe (FAM-TGGGTAGCTTTGGTGGACCGATGTTTA ATCT-BHQ1) to determine O. tsutsugamushi copy numbers [14 (link)]. All qPCRs were run in triplicate in Applied Biosystems’ QuantStudio 3 (Thermo Fischer Scientific, Waltham, MA, USA).