T4 dna ligase enzyme

The plasmids were prepared by amplifying the gene encoding the metagenomic DNA of the samples extracted for the sequences available in the LBMP⁵⁵ . The following specific primers (Sigma-Aldrich) were synthesized using the commercial PCRBIO Ultra Mix 2 × Kit^{26 (link)}: forward 5'-TATAgaattcTTCGGACGCAATCCAGACACCAATCC-3', which contains the restriction site EcoRI, and reverse 3'-TATAaagcttTTACTTGAACAGCAATTGAG-5', which contains the restriction sites EcoRI and HindIII. The fragment corresponding to the gene was purified by the Zymoclean Gel DNA Recovery Kit from ZymoResearch. The vector pET28a ( +) (INVITROGEN) and insert were restricted with restriction enzymes (FastDigest EcoRI, Thermo Scientific; and FastDigest HindIII, Thermo Scientific) and dephosphorylated with the enzyme Fast Alkaline Phosphatase (1 U/µL, Thermo Scientific). The connection between the insert and the vector was performed according to the protocol of Sambrook and Russel^{56 (link)} using the T4 DNA ligase enzyme (New England Biolabs) in a 3:1 ratio (insert: vector).

Genomic DNA was isolated using the Plant DNAeasy^® kit (Qiagen), following the manufacturer’s protocol. Genomic DNA concentration was adjusted to 20 ng/μl, and ApeKI GBS libraries were prepared following the protocol described by Eslhire et al. [35 (link)]. DNA of each sample (200 ng) was digested with the ApeKI enzyme (New England Biolabs, Hitchin, UK). Digestion took place at 75°C for 2 h and then 65°C for 20 min to inactivate the enzymes. The ligation reaction was completed in the same plate as the digestion, again using T4 DNA ligase enzyme (New England Biolabs, Hitchin, UK) at 22°C for 1 h and the ligase was inactivated prior to pooling the samples by holding it at 65°C for 20 min. Ligated samples were pooled and PCR-amplified in a single tube but further complexity reduction was achieved using PCR primers with one selective base (A) as per Sonah et al. [47 (link)]. Single-end sequencing was performed on a single lane of an Illumina HiSeq2000 (at the MGX platform in Montpellier, France).The Illumina Hiseq 2000 sequencing raw data are available in the NCBI SRA (Sequence Read Archive), under the study accession number: SRP109295.

Ion Torrent compatible-NEXTflex DNA Barcodes- (Bioo Scientific, Austin, TX), were selected as a cost-effective alternative to standard IonXpress barcodes supplied by ThermoFisher Scientific. Barcode ligation was performed with the T4 DNA ligase enzyme component of the NEBNext fast DNA library prep set for Ion Torrent (New England Biolabs, Ipswich, MA). Barcoded products were then subjected to a purification process using 180μl Agencourt AMPure XP mix (Beckman Coulter, Brea, CA) and subsequent 80% (v/v) Ethanol wash. Following this, the barcoded mtDNA fragments were examined on an E-gel system (ThermoFisher Scientific, Waltham, MA) utilising an iBase unit to visualise the running gel. This allowed us to identify and recover fragments between 250–350 base pairs. Libraries were size selected at a target peak of 330bp thus maximising efficiency under the limitations of 200bp sequencing chemistry.

The amplified PCR product was digested using NdeI and XhoI restriction enzymes and the digested product was gel purified using a QIAquick gel-extraction kit (Qiagen, Germany) and cloned into the pET22b+ expression vector (Fig. 2B) (Novagen) using T4 DNA ligase enzyme (NEB, England) and transformed into DH5alpha E. coli competent cells to increase the copy number, subsequently the recombinant vector with pfgdh gene was transformed into BL21 (DE3) E. coli cells as per manufacturers recommended protocol. The clone designated with rPfGDH the expresses the protein of interest (residues 1–470 plus 6xHis tag at C-terminal) was selected for further studies.

The ZIKV envelope (E_ZIKV) sequence (amino acids 291-690 of the ZIKV polyprotein) was generated as previously described (37 (link)). The sequences were codon optimized for prokaryote or eykaryote expression (GenScript, NJ) and cloned into pET21a plasmid using NheI and XhoI restriction sites (named as pET21a-E_ZIKV) and in pVAX (pVAX-E_ZIKV) vector using HindIII/XhoI. Then, the ectodomains EDI/II_ZIKV (aa 291-600) and EDIII_ZIKV (aa 601-690) sequences were amplified by PCR with specific primers (Supplementary Table 1) using Phusion High Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer’s instructions. The PCR products were cloned into the pJET1.2/blunt vector (Thermo Scientific) and digested with restriction enzymes NheI and XhoI (New England Biolabs) for bacteria expression and NcoI and XhoI for S2 expression. The digested fragment was purified using PureLink Quick Plasmid DNA kit (Invitrogen) and cloned in frame with the open reading frames of pET21a or pMT/BiP/V5-HisB vector using T4 DNA ligase enzyme (New England Biolabs). Plasmids were sequenced and transformed into DH5α bacteria for DNA purification using the EndoFree Plasmid Maxi Kit (Qiagen) according to the manufacturer’s instructions.

DMEM, fetal bovine serum, trypsin and penicillin–streptomycin was purchased from Life Technologies. DNA restriction enzymes and T4 DNA ligase enzyme were from New England Biolabs. Cell culture dishes were from Sarstedt AG & Co, and white 96-well plates were from Nunc. [³H]5-HT (27–28 Ci mmol⁻¹), [³H]imipramine (41 Ci mmol⁻¹), MicroScint-0 and -20 scintillation fluid and 96-well glass fibre filter plates were obtained from PerkinElmer Life Sciences. [³H]vortioxetine (69 Ci mmol⁻¹) was purchased from Ubichem Pharma Services (Hungary). azF and BzF were from Chem-Impex International, Inc. Rho1D4 mAb was obtained from The National Cell Culture Center.

The pCW-3A4-BMR plasmid containing the gene of human P450 3A4 fused to the reductase of cytochrome P450 BM3 from B. megaterium (BMR) linked via a Pro-Ser-Arg linker was available in our laboratory (Dodhia et al., 2006 (link)). GenElute plasmid miniprep kit was obtained from Sigma (Italy). PCR purification kit and gel extraction kit were purchased from Macherey-Nagel (Germany).
The primers for PCR and oligonucleotides for the linker region between P450 3A4 and BMR reductase were prepared by MWG (Germany). DNA Ladder, restriction endonucleases enzymes Avr II and Hind III, Vent polymerase, T4 DNA ligase enzyme and buffers used in sub-cloning were from New England Biolabs (UK) and Promega (Italy). Horse heart cytochrome c, erythromycin, testosterone, 6β-hydroxytestosterone, horseradish peroxidase type X, superoxide dismutase from bovine erythrocytes, catalase, N,N-dimethylaniline, and 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one were purchased from Sigma (Italy). NADPH (tetrasodium salt) was acquired from Calbiochem (Germany). Human CPR was purchased from Life Technologies. All other chemicals, biochemicals used in this study were purchased from Sigma (Italy) at the highest available grade and used as recommended by the manufacturer.