Pyrobest dna polymerase

Samples of 100 ng of purified mRNA were used for synthesis of the first cDNA strand using Reverse-transcription RT Kit (Invitrogen, Carlsbad, CA, USA). All polymerase chain reactions were performed with Pyrobest DNA Polymerase (Takara Biotech Co. Ltd) in a total volume of 25 mL reaction mixture consisting of 10 × Pyrobest buffer, 2.5 mL; 2.5 mM dNTP mixture, 2 mL; 1st strand cDNA, 2 mL; 20 mM forward primer, 0.25 mL; 20 mM reverse primer, 0.25 mL; Pyrobest DNA Polymerase, 0.25 mL; ddH₂O, 17.75 mL (Forward primer, 5′-ACGCCCTTCCTGAACAAG-3′, Reverse primer, 5′- ATCTGCTGCTGCTTCTGC − 3′). The internal reference gene was Atactin8 (Forward primer: 5′- CTATTGTCTGTGACAATGG-3′; Reverse primer: 5′- AACCCTCGTAGATAGGCA − 3′). The reaction program was as follows: 98 °C for 10 s; 55 °C for 5 s; 72 °C for 2 min, 30 cycles. The products were ligated into the T-vector (pEasy-blunt simple cloning kit, TransGen Biotech, Beijing, China) for sequencing (Shanghai Biotech Co.).

Plasmid (pET15b-Tat-GFP-Tat) expressing GFP protein with HIV-1 Tat PTD (amino acids 49–57) at both N-terminus and C-terminus was provided by Dr. Soo Young Choi at Hallym University, Korea. The gene corresponding to CTA1 subunit (amino acids 1–194) was amplified with a forward primer (5′-GGGCCCCTCGAGAATGATGATAAGTTATATCGG-3′) and a reverse primer (5′-CCCGGGGGATCCCGATGATCTTGGAGCATTCCC-3′) by polymerase chain reaction (PCR). Vibrio cholerae N16961 strain (AE003852) was used as a template for CTA1 subunit. The PCR product was digested with Xho I and BamH I and then ligated to the pET15b-Tat-GFP-Tat plasmid which was linearized with the same enzymes, resulting in the recombinant plasmid pET15b-TCTA1T. Plasmid pET15b-TmCTA1T, which has a point mutation (Ser63→Lys) at ADP-ribosyltransferase enzymatic active site of CTA1, was generated by site-directed mutagenesis with 5′-TATGTTTCCACCAAGATTAGTTTGAGA-3′ and 5′-TCTCAAACTAATCTTGGTGGAAACATA-3′ primers using Pyrobest DNA polymerase (Takara, Japan). The pET15b-TCTA1T plasmid was used as a template for TmCTA1T (Figure 1(a)), and the DNA sequences were confirmed at Macrogen (Seoul, Korea).

PCR amplification was performed using the bacterial 16S rRNA gene V5–V7 region primers 799F (5′-AAC MGG ATT AGA TAC CCK G-3′) [10 (link)] and 1193R (5′-ACG TCA TCC CCA CCT TCC-3′) [11 (link)]. Fungal rRNA internal transcribed spacer gene ITS1 was amplified with primers 5′-CTT GGT CAT TTA GAG GAA GTA A-3′ and 5′-GCT GCG TTC TTC ATC GAT GC–3′) [10 (link)]. The reverse primers were modified to contain a barcode [12 (link)]. A volume of 50 μL PCR reaction contained 5 μL 10× Pyrobest Buffer (Takara, Dalian, China), 1 μL DNA template, 2 μL of each primer (10 μmol/L), 4 μL dNTPs (2.5 μmol/L), 0.3 μL Pyrobest DNA Polymerase (2.5 U/μL, Takara, Dalian, China), and 35.7 μL ddH₂O. PCR reaction procedure for bacterial 16S rRNA gene consisted of an initial 94 °C for 3 min, followed by 28 cycles of 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s, and a final extension of 72 °C for 10 min. PCR reaction procedure for fungal rRNA gene ITS1 consisted of an initial 95 °C for 2 min followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s and a final extension of 72 °C for 5 min. PCR products were cleaned using the UltraClean PCR Clean-up Kit (Carlsbad, CA, USA).
The cleaned PCR products were passed to Beijing Fixgene Techology Co., Ltd. and sequenced using an Illumina Hiseq instrument with a PE250 paired-end (Hiseq 2500, PE250).

A tolC-expressing plasmid was constructed by cloning a PCR-amplified fragment containing the 5ʹ-untranslated and the coding regions of tolC between the SacI and HindIII sites of the vector pBAD33 to yield plasmid pKRB2100 encoding wild-type TolC. The upstream primer used was designed to cover 28 bases just upstream of the deduced Shine-Dalgarno sequence of chromosomal tolC. A derivative of pKRB2100 encoding TolC-A269C (pKRB2104) was constructed using the QuickChange II one-day site-directed mutagenesis method (Stratagene) with Pyrobest DNA Polymerase (Takara Bio) and DpnI (New England Biolabs).

The bacterial strains and vectors involved in this study are listed in Table 1. E. coli Top 10 was used as a host for the cloning and induced expression of the plasmid-coded reporter proteins. E. coli was grown in Luria Broth (OXOID, Basingstoke, UK) at 37°C. Pyrobest DNA polymerase, restriction enzymes, and gel extraction kits were purchased from TaKaRa (Dalian, China). Isopropyl-β-D-thiogalactopyranoside (IPTG), o-nitrophenyl-β-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), ampicillin, and lysozyme were obtained from Sangon Biotech (Shanghai, China). All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). All oligonucleotides were synthesized by Sangon Biotech (Shanghai, China).

After quantification with NanoDrop measurement, 30 ng of DNA was used for Polymerase Chain Reaction (PCR) amplification. The variable region of 16S rDNA V3–V4 (338–806) was used to design primers as follows: 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTA CNNGGGTATCTAAT-3′) (Munyaka et al., 2015 (link)). PCR was performed in triplicate in 25-μl volumes containing 2.5 μl of 10 × Pyrobest Buffer, 2 μl of 2.5 mM dNTPs, 1 μl of each primer (10 μM), 0.4 U of Pyrobest DNA Polymerase (TaKaRa, Dalian, China), and 15 ng of template DNA (Chen H. et al., 2021 (link)). PCR conditions were pre-set as: pre-denaturation was performed at 94°C for 3 min, followed by 25 cycles of denaturation at 94°C for 30 s, then annealed at 50°C for 30 s and extended at 72°C for 60 s (Xing et al., 2021 (link)). After this, agarose gel electrophoresis was performed to detect the specificity of the amplification results. The gel was prepared at a concentration of 1%, running at a voltage of 170 V, for 30 min.