Puc118 ecori bap vector

To create the metagenomic library of the human gut, we randomly selected stool samples from 10 Kunming University of Science and Technology students who had not used antibiotics in the past 2 years. The Ethics Committee of the First People’s Hospital of Yunnan Province approved this study, and informed consent was obtained from the individuals contributing fecal samples. Using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Catalog no. 51604), we immediately extracted DNA from the gut microbiota after sample collection, following the manufacturer’s instructions.
Next, 15 μg of metagenomic DNA was subject to digestion with the EcoRI restriction enzyme (Takara, Code No.1040S). Fragments ranging from 1 to 3.5 kb were selected through electrophoresis on a 1% agarose gel. The corresponding gel slice was excised, and DNA was extracted using a gel purification kit (Zomanbio, ZP202). The retrieved DNA was then ligated into the pUC118 EcoRI/BAP vector (Takara Code No. 3320). The subsequent procedures, including ethanol precipitation, electroporation, determination of average insert size, total library size count, and library storage, mirrored those used for constructing the soil metagenomic library. The average insert size for the gut metagenomic library was calculated to be approximately 2.0 kb, with a total library size of 1.0 Gb.

The fungal chromosomal DNA was isolated from the grinded frozen mycelium with DNeasy plant kits (Qiagen) without using QIAshredder (Qiagen) and extracted with phenol–chloroform. The DNA (0.1 mg) was partially digested with two units of MboI at 37 °C for 4 min and dephosphorylated with calf intestinal alkaline phosphatase (CIAP). The genomic DNA fragments were ligated into XbaI-digested, dephosphorylated, and BamHI-digested SuperCos1 cosmid vector (Agilent Technologies, Santa Clara, CA). Gigapack III Gold packaging extract (Agilent Technologies) was used for packaging the DNA according to the manufacturer’s instructions. E. Coli XL1-Blue MR (Agilent Technologies) was transfected by the resulting recombinant phage. Approximately 50,000 colonies from the genomic library blotted on Amersham Hybond-N+ membranes (GE Healthcare) were screened by hybridization with digoxigenin (DIG)-labeled partial necC DNA fragments using Dig Easy Hyb (Roche Diagnostics) at 43 °C for 14–16 h. The membranes were washed with 0.5 × SSC/0.1 % SDS at 55 °C. Detection was performed using CDP-Star (Roche Diagnostics). The positive cosmids were isolated, then digested with EcoRI and BamHI separately and subcloned into pUC118 EcoRI/BAP vector or pUC118 BamHI/BAP vector (Takara Bio, Shiga, Japan). The DNA fragments were sequenced as described above and assembled using Genetyx software (Genetyx, Tokyo, Japan).