Axyprep plasmid miniprep kit

All primers used in this study (Supplementary Table S6) were designed by the Primer Premier 5.0 software (http://www.premierbiosoft.com/; last accessed Jan 2019) and synthesized by the BGI Tech (Shenzhen, China). PCR was performed in a total volume of 15 μl, containing 7.5 μl of GC buffer I, 50 ng of DNA, 1 μl of forward and reverse primers (10 mM), 0.24 μl of dNTPs (25 mM), and 0.15 μl of LA Taq polymerase (Takara, Dalian). PCR was carried out in a Veriti 96-Well Thermal Cycler (Applied Biosystems, USA) with the following procedure: denaturing at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, annealing at 55–65 °C for 45 s, and 72 °C for extension (1 kb min^–1), and finally extension at 72 °C for 10 min. PCR products were purified by an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Hangzhou), cloned into the pEASY-T1 Cloning Vector, and then transformed into Trans1-T1 competent cells by the heat shock method (TransGen Biotech, Beijing). Conjugative plasmids were extracted with an AxyPrep Plasmid Miniprep Kit (Axygen Biosciences) and sequenced by an ABI 3730XI DNA Analyser (Applied Biosystems).

The conjugation experiment of biparental mating on the sterile nitrocellulose filter was performed using the multiple resistant P. aeruginosa as the donor cell and the rifampin-resistant Escherichia coli C600 (EC600) as the recipient (He et al., 2021 (link)). The transconjugants were selected on Mueller–Hinton agar plates containing 600 μg/ml rifampin and 32 μg/ml ceftazidime or 8 μg/ml meropenem. The target resistance gene in the transformant was verified by PCR with primers of the corresponding β-lactamase resistance genes (Table 1). The PCR product was sequenced and the sequence was compared with those in the public database by the BLASTN program (see footnote 15). The plasmid of the transformant was extracted with an AxyPrep Plasmid Miniprep Kit (Axygen Scientific, Union City, CA, United States), and the target resistance gene on the plasmid was also verified by PCR and PCR product sequencing as mentioned above.

To generate promoter deletion constructs, primary monocyte genomic DNA was extracted from the interphase and phenol layer using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. To obtain 5′ end promoter deletion products, primers (Supplementary Table S1) flanking the desired promoter regions were used for PCR amplification, using the purified genomic DNA as template. PCR was performed using iProof High Fidelity DNA Polymerase (Bio-rad) under the following parameters: initial denaturation at 98°C; 40 cycles of amplification, denaturation at 98°C for 10 s, annealing at Tm of primer pair +3°C for 30 s, elongation at 72°C for 2.5 min; followed by a final extension at 72°C.
The isolated promoter lengths were separately cloned into pGL4.20 vector (Promega) using standard molecular cloning techniques. The restriction enzymes used were XhoI and HindIII (Thermo Fisher Scientific). T4 DNA ligase was from Roche. For small-scale purification of plasmids, AxyPrep Plasmid Miniprep kit (Axygen Biosciences) was used. Large-scale purification of plasmids intended for transfection was carried out using PureLink HiPure Plasmid Filter Purification kit (Invitrogen). The full-length sequence of each promoter construct was confirmed by sequencing using Big Dye Terminator cycle sequencing kit and ABI Prism 3100 Genetic Analyzer (Life Technologies).

The transfected cells were lysed and gDNA was extracted using the DNeasy Tissue Kit (Qiagen) following the manufacturer’s instruction. For HDR-positive events identification, PCR was performed using PrimeSTAR HS DNA Polymerase (Takara) with sequence-specific primers (Supplementary Table S3) using the condition: 95 °C for 4 min; 35 cycles of 95 °C for 20 s, 60 °C for 30 s, 72 °C for 1 min; 72 °C for 2 min for the final extension. PCR products were run on 1.5% agarose gel (Biowest). The specific DNA bands were recovered using AxyPrep DNA Gel Extraction Kit (Axygen). Purified PCR products were cloned into the pMD-19 T vector (Takara) according to the standard manufacturer’s instructions or directly sequenced by specific primers. Plasmid mini-preperations were performed using the AxyPrep Plasmid Miniprep Kit (Axygen), and midi-preparations were performed using QIAGEN Plasmid Plus Midi Kit (Qiagen). All sequencing confirmations were carried out using Sanger sequencing.

High fidelity restriction endonucleases BamHI, EcoRI, HindIII, SalI, XhoI, and T4 ligase were purchased from NEB (New England Biolabs, MA, U.S.A.). Taq DNA polymerase and PFU DNA polymerase were purchased from TransGen (TransGen Biotech, Beijing, China). Bacterial strain Pro 5-α was purchased from Promega (WI, U.S.A.). Plasmids were prepared by AxyPrep™ Plasmid Miniprep Kit (Axygen, Corning, MA, U.S.A.). DNA products were purified by AxyPrep™ PCR Clean-up Kit (Axygen). Restriction enzyme digested-fragments were extracted by AxyPrep™ DNA Gel Extraction Kit (Axygen). DFHBI-1T was purchased from MCE (NJ, U.S.A.). DAPI was purchased from Solarbio (Beijing, China). HeLa and NIH-3T3 were gifts from Prof. Wang Fei’s laboratory (Chinese Academy of Sceinces, Chengdu, P.R. China).

Real-time PCR was conducted to estimate the abundance of bacterial and fungal species. The reactions were based on 2× TaKaRa SYBR^® Premix Taq™ (Bio technology Dalian Co. Ltd., Dalian, China) using a Mastercycler ep realplex 2S device (Eppendorf, Germany). The primer pairs were Eub338/Eub518 (bacteria) and ITS1-F/ITS2 (fungi). Each 25 μL reaction contained 10 µL 2× SYBR^® Premix ExTaq™, 0.5 µL of each primer, 0.5 μL 50× ROX Reference Dye II and 2 µL DNA. For the bacterial assay, the PCR comprised an initial denaturation of 95 °C/10 min, followed by 40 cycles of 95 °C/10 s, 54 °C/10 s, 72 °C/30 s, while for the fungal amplicons, the initial denaturation step was 95 °C/5 min and the cycling regime was 45 cycles of 95 °C/15 s, 53 °C/30 s, 72 °C/45 s. Standard curves were generated using a 10-fold serial dilution (from 10⁹ to 10⁴ copies) of a plasmid containing a full length copy of Saccharomyces cerevisiae 18S rRNA or Escherichia coli 16S rRNA [3 (link),14 (link)]. Plasmid was extracted by Axyprep™ Plasmid Miniprep Kit (Axygen, Hangzhou, China). Each biological sample was subjected to three technical replicates.

Chromosomal DNA was isolated by phenol-chloroform extraction. Plasmid DNA was isolated and purified using AxyPrep Plasmid Miniprep Kit or AxyPrep Plasmid Midiprep Kit (Axygen Biosciences, USA) according to the manufacturer's instructions. PCR was carried out with Taq polymerase with initial denaturation of 2 min at 94°C followed by 30 cycles of amplification steps of 30 sec at 94°C, 1 min at 52°C, and 1 min at 72°C. PCR products were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences). DNA sequencing was performed employing ABI 3100 automated DNA sequencer using the Big-Dye Terminator Kit (Applied Biosystems, USA).