Ecori restriction enzyme

The recombinant plasmid GpBSK-BEV-E0 was linearized by the EcoRI restriction enzyme (Takara, Beijing, China). The enzyme digestion system comprised 10 μg recombinant plasmid, 40 μL of 5× CutSmart buffer, 8 μL EcoR I-HF, and RNase Free ddH₂O up to 200 μL. Enzyme digestion was performed at 37 °C for 3 h, and the restriction enzyme was inactivated at 65 °C for 20 min. Linear plasmids were recovered from the agarose gel and purified by ethanol precipitation.

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.