Premix taqtm

To express Fh3GT1 in E. coli, the full length cDNA of Fh3GT1 was amplified by PCR using Premix Taq^TM (TaKaRa) with the primers PF2 and PR2 (Supplementary Table S1). The PCR product was sub-cloned into the pET-28a (+) His-fusion protein expression vector, and clone authenticity was confirmed by sequencing. The constructed vector was transformed into E. coli BL-21 (DE3) cells for recombinant protein expression. Then, the transformants were pre-cultured at 37°C for 12–14 h in Luria Broth (LB) media containing 100 mg L^-1 kanamycin. Two milliliter of the preculture was transferred to the fresh LB media (400 ml) containing the antibiotics and grown at 37°C until an A₆₀₀ of 0.6 was reached. After the addition of 40 μl of 1 M isopropyl-β-d-thiogalactopyranoside (IPTG), the induced culture was further incubated at 16°C for 50 h. The cells were harvested by centrifugation, resuspended in 20 ml of phosphate-buffered saline (PBS, pH 7.4), and disrupted by sonication. After centrifugation at 12,000 rpm for 20 min, the supernatant was applied to a column containing 3 ml Ni Sepharose (GE Healthcare) that had been equilibrated with PBS. Bound protein was eluted from the column using 100 mM imidazole in PBS. The purified proteins were collected and analyzed on SDS-PAGE. Expressed proteins in the gels were stained with Coomassie brilliant blue R-250 for visualization.

The PCR amplification of the full-length 16S rRNA gene for SMRT sequencing was carried out by using the amplification program of: 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 55°C 30 s, and 72°C for 1 min, with final elongation of 72°C for 5 min. Reaction was performed in a 25 μL amplification mixtures with 12.5 μL Premix Taq^TM (TaKaRaTaq^TM Version 2.0, Takara Biotechnology), 0.5 μL of forward primer 27F (5’-GAGAGTTTGATCCTGGCTCAG-3’), 0.5 μL of reverse primer 1492R (5’-TACCTTGTTACGACTT-3’), 1 μL of DNA, and 10.5 μL of PCR-grade water. The quality control for PCR amplifications and sequences pre-processing were performed as described by Mosher et al. (2013) (link). The 16S rRNA library was built with a Pacific Biosciences Template Prep Kit. Sequencing of the amplicons was performed on a PacBio Sequel instrument (Pacific Biosciences, Menlo Park, CA, United States). The SMRT sequencing and sequencing data analysis work were carried out by the company of Macrogen, Shengzhen sector, China, in year 2016.

Total RNA was isolated with TRIZOL reagent (Invitrogen, CA, United States) according to the manufacturer’s instructions and subjected to reverse transcription reactions using PrimeScript RT reagent kit (Takara, Dalian, China). The quantitative PCR analysis was carried out using the CFX96 Real-Time PCR Detection System (Bio-Rad, CA, United States) with SYBR premix Ex Taq II kit (Takara, Dalian, China) according to the manufacturer’s instructions. Programs for real-time PCR are as follows: 95°C for 30 s, 95°C for 5 s, and 60°C for 30 s, repeating 40 cycles. Gapdh was used as a reference gene. The melting curves did not detect any non-specific amplification. All sample values were normalized to Gapdh expression by using the 2^–△△Ct method; primer efficiency correction was done and used to corrected amplification efficiency, Gradient concentration DNA samples were used for the normalization. The PCR primer sequences are listed in Table 1.
The semiquantitative PCR was preformed using premix Taq^TM (Takara, Dalian, China) kit, with the following programs: 92°C for 3 min for one cycle; 92°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 35 cycles. Then, PCR products were electrophoresed on 1% agarose gels (Invitrogen, CA, United States) and visualized by UV light.