E z n a plasmid mini kit

Standardized cloning procedures such as PCR and DNA restrictions were carried out according to Sambrook and Russell, 2001. Plasmids were isolated from 5 mL liquid cultures using the E.Z.N.A.^® Plasmid Mini Kit (Omega Bio‐tek, Inc., Norcross, USA) following manufacturer's instructions. PCR fragments were purified with the NucleoSpin^® Gel and PCR Clean‐up Kit (Macherey‐Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer's instructions. Chromosomal DNA of E. coli MG1655, P. putida, C. maltaromaticum, and L. lactis was isolated using the Nucleospin^® Microbial DNA Kit (Macherey‐Nagel GmbH & Co. KG, Düren, Germany) following the protocol of the manufacturer. Electrocompetent cells were prepared for E. coli and P. putida as described previously 53, 54. E. coli DH5α and P. putida strains were electroporated with an Eporator (Eppendorf AG, Hamburg, Germany) at 2.5 kV with 600 Ω resistance. All enzymes for recombinant DNA work were obtained from Thermo Fisher Scientific Inc. (Darmstadt, Germany) and oligonucleotides were synthesized by biomers.net GmbH (Ulm, Germany, listed in Table 3).

The DNA sequence of NTPase-II gene was amplified from the construct pBAD-HisB-NTPase-II (Tan et al., 2011 (link)) using a set of specific primers 5′-TTCCCGGGATGACAGACTCATCGTCACTCC-3′ and 5′-CCACTAGTTCACAGATTGTGAGAATATCCCGCC-3′ and was inserted into Xma I and Spe I site of pRREP-eGFP (Karlsson and Liljestrom, 2004 (link)) to swap the coding sequence of eGFP. The resulting plasmid, pRREP-NTPase-II, was purified with E.Z.N.A.^® Plasmid Mini Kit (OMEGA Bio-tek, USA) and stored at -20°C. Prior to immunization, the plasmids were linearized and RNAs in vitro transcription were made by using MEGAScript^TM Kit (Ambion, USA) as described previously (Geall et al., 2012 (link)). Finally, the purified transcribed RNAs were capped with Vaccinia Capping System (NEB, USA) before encapsulation (Figure 1A).

The pEASY-E1 vector was used for high level, inducible expression of ZmCAT recombinant proteins, as has been reported by Liu et al. (2016) (link). In brief, the cDNA fragment encoding the mature peptide of ZmCAT was obtained after PCR amplification with the primers, ZmCAT-recombinant-F and ZmCAT-recombinant-R (Table S1). The plasmid pEASY-E1/ZmCAT was isolated by E.Z.N.A.^®  Plasmid Mini Kit (Omega Bio-Tek Inc., Norcross, GA, USA) and then transformed into E. coli strain BL21 (DE3) to produce recombinant protein. The resulting strain was grown at 37 °C in liquid SOB medium coupled with ampicillin (100 mg  mL⁻¹) until the OD₆₀₀ reached 0.4–0.6. The bacteria were induced for protein expression by IPTG addition to 1 mM. After incubation for 4 h at 30 °C, the bacteria were harvested by centrifugation and the recombinant protein purified by MagExtractorTM His Tag Kit (Toyobo Co Ltd., Osaka, Japan) under denaturing conditions (8 mol L⁻¹ urea). The purified protein was next refolded in a gradient urea-TBS glycerol buffer and the resultant protein mixed 1/1 (wt/wt) with 2× Coomassie blue loading dye. Each sample was loaded on a 12% SDS-PAGE gel and Precision Plus Protein™ standards (Bio-Rad Laboratories Inc., Hercules, CA, USA) added to provide molecular mass markers.

We used taxon-specific primers (either previously published or designed for this study) on a subset of tick samples positive for Rickettsia, Rickettsiella, Borrelia, and Cardinium (Table 2 and Table 3). We also assayed the pooled DNA with PCR specific to Wolbachia, nematode or chalcid wasp genes (Table 2 and Table 3) but did not sequence these amplicons. Amplicons were separated on a 0.5X TAE-2% agarose gel. Bands were excised and purified for cloning with the StrataClone PCR Cloning kit (#240205, Agilent, Santa Clara, CA, USA), following manufacturer’s guidelines. Plasmids were purified using the E.Z.N.A. Plasmid mini kit (Omega Biotek #D6942) and sequenced in both directions on an Applied Biosystems (ABI) 3130/Genetic Analyzer. Sequences were compared to known sequences in the Genbank NR nucleotide database.
Sequences were aligned, trimmed, and phylogenetically analyzed in MEGA7 [34 (link)]. The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model [35 ]. Bootstrap consensus was inferred from 1000 replicates and branches in fewer than 50% bootstrap replicates were collapsed [36 (link)]. Genbank accession numbers used for each taxon/gene analysis are listed in Table 4.

The pden_1323 gene from P. denitrificans PD1222 was codon-optimized for expression in E. coli with NdeI and XhoI restriction sites included at the 5′ and 3′ ends, respectively. The vector containing pden_1323, as well as an empty pET-28b(+) vector, was digested with these restriction enzymes. The pden_1323 product was purified via a Lonza FlashGel DNA Cassette and recovered using an Omega Bio-tek E.Z.N.A. Gel Extraction Kit, while the linearized pET-28b(+) vector was purified using an Omega Bio-tek E.Z.N.A. Cycle Pure Kit. The pden_1323 insert and the digested pET-28b(+) vector were then ligated together using T4 DNA ligase using the manufacturer's protocol. The ligated mixture was then transformed into E. coli TOP10 cells and plated on Luria–Bertani (LB) agar plates containing 50 μg/ml kanamycin. Colonies were picked, grown in liquid LB medium, and plasmid preparations were made using the Omega Bio-tek E.Z.N.A. Plasmid Mini Kit.