Molecular imager gel doc xr system

Genomic DNA extraction used the Quick-DNA Fungal/Bacterial Microprep Kit (Zymo Research, Irvine, US) and followed the manufacturer’s instructions. DNA concentrations were quantified with a Nanodrop One spectrophotometer (Thermo Scientific, Waltham, US), and DNA integrity was assessed on agarose gel (1%). Amplification of the 16 S rDNA gene used the universal primer pair 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) [35 (link)]. The PCR was performed on a CFX96 Real-Time System thermal cycler (Bio-Rad, Hercules, US) using the following protocol: Initial denaturation for 5 min at 95 °C, followed by 30 cycles of 40 s at 95 °C for denaturation, 50 s at 60 °C for annealing, 40 s at 72 °C for extension, and 10 min at 72 °C for a final extension. The amplified products were visualized by electrophoresis on agarose gel (1%) using the Biorad Molecular Imager Gel Doc XR + System (Bio-Rad, Hercules, US) (Fig. S2). The amplicons were sequenced by the Sanger technique at the Institute of Biotechnology of the National Autonomous University of Mexico (UNAM, Mexico City).

Integration site analysis was performed by LM-PCR as reported before^{11 (link),58 (link)}. Genomic DNA was digested with CviQI restriction enzyme for MRV and with HaeIII for MRV* pre-selected H9M-ciMPs (New England BioLabs, Frankfurt/ Main, Germany). To amplify integration sites of MRV vectors, 5′ LM-PCR was performed using the following primers Rv1 5′-[bio] ACGTTCTCTATCACTGATAGGGAGTAAACTGG-3′; Rv2, 5′-CAGGCACAAGTGTTAAAGCAG-3′; Rv3, 5′-TCGAATTCACGTGTCGACGA-3′. To amplify integration sites of MRV* vectors, 3′ LM-PCR was performed using a primer that annealed in the wPRE sequence (SINPRE, 5′-[bio]GCACTGATAATTCCGTGGTGTTGTC-3′). For exponential PCR, the following primers were designed: SIN LTR2_1, 5′-GTTTGGCAAGCTAGCGAGACT-3′ and/or SIN LTR2_2, 5′-CCCTATCAGTGATAGAGAACGT-3′; SIN LTR3, 5′-CCCAATAAAGCCTCTTGCTGT-3′. PCR products were extracted after gel electrophoresis using QIA quick Gel Extraction Kit (Qiagen), sequenced and analysed using BLAST/BLAT searches at www.ensembl.org/Mus_musculus/Tools/Blast42 (Ensembl release 100 April 2020)^{59 (link)}. LM-PCR for H9M-ciMP #38 did not reveal any specific bands. The images of full-length gels presented in Supplementary Figure S1, S9. The photos were taken using BIO-RAD Molecular Imager Gel Doc XR + System with Image Lab Software (Bio-Rad Laboratories, Inc. Hercules, California, USA).

Cells were lysed with RIPA lysis buffer containing protease inhibitors (#693132001, Thermo Fisher Scientific, Waltham, MA, USA) to extract protein. Protein concentrations were measured using a bicinchoninic acid (BCA) assay kit (#23227, Thermo Fisher Scientific). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Polyvinylidene fluoride (PVDF) membranes were used to transfer proteins (60–70 min) and blocked with 5% skimmed milk. The specific primary antibody (1:1000) was incubated with the PVDF membrane at 4 °C overnight. HRP-conjugated secondary antibodies (1:100,000) were then added. Protein expression was detected using the Molecular Imager^® Gel Doc™ XR System (Bio-Rad, Hercules, CA, USA) with the Enhanced Chemiluminescence (ECL) system (#P10300, New Cell & Molecular Biotech, Hong Kong, China).

The designed B22R gene fragment was codon-optimized, and the synthetic gene was cloned into the pET28a+ expression vector (Twist Biosciences, South San Francisco, CA, USA) and expressed as described below. Induction was performed in BL21 (DE3) cells with 0.1 mM of IPTG and 0.3% L-arabinose. After cellular lysis, the supernatants were harvested by centrifugation at 6000× g for 20 min and purified using fast-flow crude his-trap columns (Cytiva, MA, USA) according to the manufacturer’s instructions. Additional purification was performed by size exclusion chromatography, Superdex Increase 75 10/300 (Cytiva, MA, USA). All the purification steps were performed with an ÄKTA-Go protein purification system (Cytiva, MA, USA). The expression of the protein was confirmed by SDS-PAGE and Western blot using an HRP-conjugated anti-his-tag antibody (dilution 1 in 1000; Merck KGaA, Darmstadt, Germany) and undiluted hyperimmune sheeppox positive serum [35 (link)]. Reactive bands were visualized using the ECL Prime Western-blot-Detection reagent (Cytiva, MA, USA) and detected by the Molecular Imager Gel Doc XR System (BioRad, CA, USA).

The mixtures of 50 µg urease crude (0.08 urease units) in 100 µL EDTA-sodium phosphate buffer and DCL (0.1–10 mM) were preincubated at 37°C for 1 h. The samples were mixed with 5× sample buffer containing sodium dodecyl sulfate (SDS), heated in 10 min, and then loaded onto SDS-PAGE polyacrylamide gels 12% (w/v). After electrophoresis at 120 V for 2 h, proteins from gels were transferred onto a Pall Corporation polyvinyl difluoride membrane (Pensacola, FL) [28] (link). The membrane was blocked with phosphate-buffered saline (PBS) containing 5% (v/v) skimmed milk for 2 h at room temperature. The membrane was then incubated overnight at 4°C with 1∶2000 dilution of Abcam duck polyclonal to H. pylori urease (Cambridge, MA). After being washed with PBS three times, the membrane was further incubated for 2 h with Abcam rabbit polyclonal secondary antibody to Abcam chicken IgY-H&L diluted 1∶4000. Finally, after several washings with PBS containing 0.5% Tween-20 (v/v), the blots were developed using an Amersham Biosciences ECL Western blotting detection reagent (Buckinghamshire, UK) and immediately exposed to an AGFA CP-PU X-ray film (Mortsel, Antwerp, Belgium) for 2–10 min at room temperature. Western blot results were analyzed using a Bio-Rad Molecular Imager Gel Doc XR system (Hercules, CA). EGCG served as a reference standard and was similarly prepared.