Zymoclean gel dna recovery kit

For in vitro treatment with methylase SssI, cloned pGL3-constructs were redigested with the NheI and XhoI restriction enzymes, and then purified using the Zymoclean^TM Gel DNA Recovery Kit (Zymo Research). Purified insert fragments were subjected to in vitro methylation by methylase SssI in the presence of the methyl group donor, S-adenosylmethionine, and monitored by digestion with the SmaI enzyme. Methylated insert fragments were subsequently purified using DNA Clean & Concentrator^TM-5 Kit (Zymo Research) and ligated with digested pGL3-basic vector. The ligation mixture was directly transfected into cells. For transfections, HEK293T cells were plated into 6-well plates at a density of 2 × 10⁵ per well, and luciferase constructs (2 μg of reporter gene plasmid and 50 ng of phRL-CMV normalizing plasmid) were transfected into the cells 1 day after seeding using FuGENE® HD transfection reagent (Roche), according to the manufacturer’s instructions. After transfection for 48 h, luciferase activity was measured by the Dual Luciferase Assay system (Promega) and relative luciferase activity was calculated by normalization to the Renilla luciferase activity of the phRL-CMV plasmid. All transfection experiments were carried out in duplicate and repeated at least three times.

Plasmid minipreps were carried out using the New England BioLabs miniprep kit (NEB, Ipswich, MA, USA). Microbial genomic DNA was extracted employing the SigmaElute^TM bacterial genomic DNA kit (Sigma, St. Louis, MO, USA). For cloning, DNA was amplified by PCR using Phusion High-Fidelity DNA polymerase from New England BioLabs in 50 µl reactions under recommended conditions. Restriction enzymes and NEBuilder Hifi DNA assembly master mix were purchased from NEB and reactions were set up according to the manufacturers’ protocol. The Zymoclean^TM gel DNA recovery kit (Zymo, Irvine, CA, USA) was used to extract gel purified linearized DNA which was subsequently used for cloning.
Chemical competent E. coli were prepared and transformed by heat shock as previously described⁵⁴ . Electrocompetent C. necator were prepared and transformed as reported by Ausubel et al.⁵⁵ .

To construct porcine COL1A1 promoter vectors of different lengths, genomic DNA was extracted from porcine embryonic fibroblast (PEF) cells using the TIANapm Genomic DNA Kit (Tiangen, Beijing, China) and used as a template for polymerase chain reaction (PCR). Five promoters of pig COL1A1 with different lengths were obtained. As a positive control, the cytomegalovirus (CMV) promoter was also cloned from the pX458 vector using PCR. The promoters included 334 bp (−294–0 bp, using primers P-294-1F and P-1R), 498 bp (−458–0 bp, using primers P-458-1F and P-1R), 905 bp (−865–0 bp, using primers P-865-1F and P-1R), 1478 bp (−1438–0 bp, using primers P-1438-1F and P-1R), 2445 bp (−2405–0 bp, using primers P-2405-1F and P-1R), and 548 bp (−508–0 bp, using primers CMV-F and CMV-R). The primer sequences are shown in Table 2. PCR was performed using a KOD FX high-fidelity enzyme system (Toyobo, Shanghai, China) under the following conditions: 94 °C for 2 min; 36 cycles at 94 °C for 10 s, 60 °C for 30 s, and 68 °C for 1 kb/min (time varied depending on the fragment length); 68 °C for 2 min; and 4 °C holds. All PCR products contained a 40 bp homologous arm sequence for seamless cloning. The PCR products were purified and recovered according to the instructions provided in the Zymoclean^TM Gel DNA Recovery Kit (ZYMO RESEARCH, Irvine, CA, USA).

Escherichia coli DH5α (Invitrogen, Schwerte, Germany) was used for cloning, and E. coli BL21 (DE3) (Invitrogen, Schwerte, Germany) was used for protein expression. pET26b (+) vector was purchased from Novagen (Darmstadt, Germany).
Luria-Bertani (LB) media consisted of: 0.5% (w/v) NaCl, 1% (w/v) peptone, and 0.5% (w/v) yeast extract, and sterilized by autoclaving. Agar was added for preparation of standard agar media at 1.5% (w/v). Lysis buffer consisted of: 50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 7.8.
Nco1 and Xho1 restriction enzymes, shrimp alkaline phosphatase, and phusion high-fidelity DNA polymerase were purchased from New England Biolabs (Massachusetts, USA). Trizol^® reagent, T4 DNA ligase, Thermo Scientific RevertAid H Minus first strand cDNA synthesis kit, and PageRuler unstained protein ladder were purchased from Thermo Fisher Scientific (Ohio, USA). Zyppy^TM plasmid miniprep kit, used for plasmid isolation, Zymoclean^TM Gel DNA recovery kit, used for DNA purification, and Mix and Go E. coli transformation kit were purchased from Zymo Research (Irvine, California, USA). HyperLadder^TM 1 kb was purchased from Bioline (London, UK). Protein concentration was determined using Quick Start^TM Bradford protein assay kit from Bio-Rad (Washington, USA).

The plasmids used in this work, their main characteristics and construction details, are described in S1 Table. The DNA primers used in their construction are listed in S2 Table. Plasmids were constructed and purified using standard molecular biology procedures with proof-reading Phusion DNA polymerase (Thermo Fisher Scientific), restriction enzymes (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DreamTaq DNA polymerase (Thermo Fisher Scientific), DNA clean & concentrator^TM-5 kit and Zymoclean^TM gel DNA recovery kit (Zymo Research), and GeneElute Plasmid Miniprep kit (Sigma Aldrich) or NZYMidiprep kit (NZYtech), according to the instructions of the manufacturers. The backbone plasmids used in this work were pGEX-4T-2 (GE Healthcare) and pMal-c (New England Biolabs), for recombinant protein purification, and pEGFP-C1 (Clontech) for transfection of mammalian cells. Furthermore, pSVP247 (S1 Fig and S1 Table), a derivative of p2TK2-SW2 [28 (link)], was the backbone to generate C. trachomatis expression plasmids bearing genes whose transcription is halted by the incD terminator (T_incD) and encoding proteins with a double hemagglutinin epitope tag (2HA) at their C-terminus. The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was confirmed by DNA sequencing.