Gel recovery kit

The whole-genome DNA extracted from B. longum 020402 was used as a template, and two pairs of primers were designed to clone the target gene. The gene encoding a protein without signal peptide (34–426 amino acids) was cloned by using primer pair 1 (F: 5′-AAAAAACATATGGTGGCGGATATGCAGGGCATTG-3′; R: 5′-GTGGTGCTCGAGTCGGTAGTGCAGCACTTCGCCGGG-3′). The gene encoding GH25 LysA-like domain (34–234 amino acids) was amplified by using primer pair 2 (F: 5′-AATTATCCATGGCAGTGGCGGATATGCAGGGCATTG-3′; R: 5′-GTGGTGCTCGAGGTCGCCCTGCGCGTACGCGTC-3′). The resulting gene fragment and plasmid pET28a (+) were digested with restriction endonucleases (NEB, Ipswich, MA, USA), and then the target fragment was recovered by using purification kit (Omega bio-tek, Norcross, GA, USA) and gel recovery kit (Omega bio-tek, Norcross, GA, USA). After that, the target fragment and the plasmid were ligated by using T4 ligase (NEB, Ipswich, MA, USA) and subsequently transformed into Escherichia coli (E. coli) DH5α (Sangon Biotech, Shanghai, China). The positive colons were screened by amplification with primer pair (T7: 5′-TAATACGACTCACTATAGGG-3′; T7-Term: 5′-GCTAGTTATTGCTCAGCGG-3′) and Sanger sequencing. If the target gene had no base mutations, the vector of pET-28a (+)/target gene was constructed successfully.

The corresponding primers listed in Supplementary Table 1 were used to amplify the cbbL and cbbM genes by PCR. The PCR reaction system was 30 μL including 3 μL template DNA, 25 μL 2 × Premix Taq, 1 μL 10 mM forward primer, and 1 μL 10 mM reverse primer. The reaction conditions were as follows: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C (cbbL) or 57°C (cbbM) for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR amplification products were detected by 2% agarose gel electrophoresis, and the target fragments were cut and recovered using a gel recovery kit (Omega Bio-tek, Norcross, GA, United States). The recovered target gene fragments were sequenced on an Illumina MiSeq 300 platform by Personal Biotechnology Co., Ltd. (Shanghai, China).

The following experimental groups were used: NC group (empty vector, pcDNA3.1); pcDNA3.1 + WT1 group (including pcDNA3.1 + WT1 full sequence); pcDNA3.1 + XIAP group (pcDNA3.1 + XIAP full sequence); and pcDNA3.1 + BMI1 group (pcDNA3.1 + BMI1 full sequence). Total RNA was extracted, cDNA obtained by reverse transcription was amplified by polymerase chain reaction (PCR), and the target fragment was recovered using a Gel Recovery Kit (Omega Bio-tek, Norcross, GA). The pcDNA3.1 plasmid and target fragment from gel recovery were ligated using T4 ligase (Qiagen, Hilden, Germany) at 22°C for 3 h. Three single colonies were selected for shake culture, and the positive clones identified by enzyme digestion were sequenced.