Dna fragment purification kit

PCR reagents and primers, including Taq DNA polymerase and a DNA fragment purification kit, were purchased from Takara Biotechnology Co., Ltd.
The D-loop region of the mitochondrial gene was located at mtDNA np16028-577 and two pairs of primers, with overlapping product regions, were used to amplify the genes of the entire D-loop region. The sequences and amplified products of the two pairs of primers are shown in Table I. In addition, when designing the two pairs of primers, one universal M13 forward primer, TGTAAAACGACGGCCAGT and one reverse primer, CAGGAAACAGCTATGACC, were synthesized at the 5′ end. The PCR conditions were as follows: 35 cycles of predenaturation at 94°C for 5 min, denaturation at 94°C for 30 sec, annealing at 59°C for 45 sec and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. Next, 3 μl PCR product was mixed with 1 μl 6× loading buffer and the mixture was added into a gel well. A 2,000 bp DNA marker (3 μl) was then added and electrophoresis was performed at a voltage of 5 V/cm for 30 min. The gel was photographed using an imager camera and the DNA fragment purification kit was used for purification, according to the manufacturer’s instructions.

Insect DNA was isolated using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit (Takara, Shiga, Japan). PCR primers used a universal primer pair for the 16S rDNA V4 region (F: 5′-AYTGGGYDTAAAGNG-3′ and R: 5′-TACNVGGGTATCTAATCC-3′) and a fusion primer pair (F: 5′-index+AYTGGGYDTAAAGNG-3′ and R: 5′-TACNVGGGTATCTAATCC-3′). PCR products were purified using the TaKaRa DNA fragment purification kit. The interaction of 3′–5′ exonuclease and polymerase repairs the DNA fragment with the protruding ends. The products then add a base to the 3′ end of the DNA and assemble the junction. Enrich the DNA library fragment and test it using the method of Pico green and FLUORO to ensure quality. Libraries were then deep sequenced using Illumina Hiseq2000 according to the manufacturer’s instructions.

DNA from Pseudomonas putida YC-AE1 was extracted using Bacterial Genomic Extraction Kit (Takara, Japan). Gene bisdB (CYP450) was amplified by using bisdB-F, bisdB-R primers, while bisdAB (ferredoxin and CYP450) was amplified by bisdAB-F and bisdAB-R (Table 1). The PCR products of bisdB and bisdAB were purified using DNA fragment purification kit (Takara, Japan). The purified products were ligated to the digested pET32a( +) vector to obtain the recombinant plasmids pET-32a-bisdB and pET-32a-bisdAB. Two recombinant plasmids were transformed separately into E. coli BL21(DE3) by heat shock method. To detect the ability of transformed cells to degrade BPA, the recombinants E. coli BL21(DE3) were grown in L.B medium supplemented with BPA (100 mg l⁻¹) until (O.D 0.8) followed by induction with Isopropyl β- d-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mmol. BPA degradation was detected using HPLC, wild type E. coli BL21(DE3) with an empty vector was used as control.

The genes of GmTrx2 and GmTrx-like1 were obtained from G. molesta transcriptome. The full-length cDNA sequences of both Trx genes were amplified with specific primers (Supplementary Table S1) and G. molesta cDNA. According to the manufacturer’s instructions of the DNA fragment purification kit (TaKaRa, Kyoto, Japan) and the pMD^TM18-T Vector Cloning Kit (TaKaRa, Kyoto, Japan), the PCR products were sequentially purified and cloned into the PMD 18-T vector. The recombinant plasmid extracted was sequenced by Sangon Biotechnology Co., Ltd. (Shanghai, China).
The online bioinformatics ProtParam tool¹ was used to analyze the physicochemical properties of GmTrx2 and GmTrx-like1. The related homologous protein sequences from various species were obtained from NCBI database and analyzed using DNAman 6.0.3 software. The conserved domains in GmTrx2 and GmTrx-like1 were detected using bioinformatics tools available on the NCBI server². The phylogenetic trees based on amino acid sequence were constructed by using the neighbor-joining method with poisson model, uniform rates, and complete deletion in MEGA 5.10 software. To ensure the accuracy of the tree structure, the tree was created with 1000 replicates.

The genomic DNA of strain YC-JY1 was extracted using a bacterial genomic DNA extraction kit (TaKaRa, Dalian, China). Based on genome sequence analysis of strain YC-JY1, potential BPA degradation genes bisdA and bisdB were proposed. Gene bisdB was amplified with primers bisdB-F and bisdB-R. The bisdAB fragment was amplified from strain YC-JY1 using primers bisdAB-F, bisdAB-R (Table S2). The PCR products were purified using a DNA fragment purification kit (TaKaRa) and ligated into the pET28a(+) vectors after digestion to obtain the plasmids pET28a-bisdB and pET28a-bisdAB. The plasmids were then transformed into E. coli BL21(DE3). BPA transformation products by E. coli (pET28a-bisdAB) were analyzed using HPLC-QTOF-MS/MS.

Two subtractive cDNA libraries were prepared. The forward library (named the NT subtractive cDNA library) was prepared using NT strain mRNA as the tester and the SH strain mRNA as the driver. The reverse library (named the SH subtractive cDNA library) was prepared using SH strain mRNA as the tester and NT strain mRNA as the driver. The subtractive cDNA libraries were constructed using the PCR-Select cDNA subtraction kit (Clontech Laboratories, Inc., Mountain View, CA, USA) based on the manufacturer’s instructions. Briefly, 2 μg of mRNA was used to synthesize double-stranded cDNA. After RsaI digestion and ligation of adaptors, differentially expressed cDNAs were normalized and enriched through two rounds of hybridization and PCR amplification. The PCR products were purified using the DNA Fragment Purification Kit (TaKaRa Bio, Inc., Shinga, Japan) and then directly inserted into T/A clone vectors using the pGEM-T Easy Vector System (Promega Corp., Madison, WI, USA), transformed into chemically competent DH5α Escherichia coli cells (Invitrogen), and cultured on Luria broth media plates supplemented with ampicillin and X-Gal/isopropyl β-D-1-thiogalactopyranoside at 37°C overnight.

Genomic DNA from fecal samples (50–100 mg) was extracted using a FastDNA SPIN kit for soil (MP Biomedicals, CA, USA). The concentrations and purity of the resultant DNA were determined by a NanoDrop (NanoDrop ND-2000, USA) and stored at − 80 °C for further analysis.
The 16S rRNA gene in the fecal DNA samples was amplified by polymerase chain reaction (PCR) with primers 16s-F (5′-AGAGTTTGATYMTGGCTCAG-3′) and 16s-R (5′-TGCTGCCTCCCG TAGGAGT-3′) targeting the hypervariable V1–V2 region of the 16S rRNA gene of bacteria. PCR products were purified using a DNA Fragment Purification Kit (Takara, Japan), and then barcoded and pooled to construct the sequencing library. An Illumina Mi-seq (Illumina, USA) was used for sequencing to generate 150,150 reads. The sequences obtained were deposited in a NCBI Sequence Read Archive under accession number PRJNA327732.