One shot top10 chemically competent escherichia coli

Bacterial strains included One Shot^® TOP10 Chemically Competent Escherichia coli (Thermo Fisher Scientific) for cloning purposes, and Agrobacterium tumefaciens EHA105 (Hood et al., 1993 (link)) carrying both the binary vector of interest and the ternary plasmid pBBR1MCS-5 with a constitutive virG gene (van der Fits et al., 2000 (link)) for plant transformation.
The experiments were performed on two genotypes: the apple ‘Gala’ and the pear ‘Conference’. In vitro proliferating shoot cultures of the apple ‘Gala’ were micropropagated on Murashige and Skoog (1962) (link) medium supplemented with 0.5 mg/l 6-benzyladenine (BA) and 0.1 mg/l 3-indolebutyric acid (IBA). Cultures of the pear ‘Conference’ were micropropagated as described by Leblay et al. (1991) on a derivative of Lepoivre’s medium supplemented with 0.5 mg/l 6-BA and 0.1 mg/l IBA. All cultures were grown at 22–24° C with a 16:8 h light:dark photoperiod (cool white fluorescent tubes, 40–60 μmol m^-2 s^-1) and transferred to fresh medium every 4 weeks.

The full-length WDR11 complementary DNA in pcDNA–green fluorescent protein (GFP) or pcDEST-Myc vector was mutagenized by using a Q5 site-directed mutagenesis kit (New England BioLabs, Ipswich, MA) to introduce the c.1306A>G variant, following the manufacturer’s protocol. Briefly, the nucleotide exchange was introduced by using Q5 Hot Start High-Fidelity DNA Polymerase with “nonoverlapping” mutagenic primers (NEBase Changer; New England BioLabs) via polymerase chain reaction run for 25 cycles of 98°C for 30 seconds, 98°C for 10 seconds, 60°C for 30 seconds, 72°C for 5 minutes, and 72°C for 2 minutes. The products were ligated at room temperature for 5 minutes and transformed into Top10 chemically competent cells [One Shot Top10 chemically competent Escherichia coli (Thermo Fisher Scientific, Hertfordshire, United Kingdom)]. Mutated plasmid constructs were verified by sequencing (Source Bioscience, Nottingham, United Kingdom). Sequences of primers used are available upon request.

The IL33 promoter and enhancer sequences were PCR‐amplified and subsequently extended with In‐Fusion primers and gel‐purified (Table 1A). pGL4.10 vector (Promega, Madison, WI, USA) was digested with HindIII and gel‐purified. The pGL4.10‐IL33‐promoter plasmid was constructed by In‐Fusion cloning (Clontech, Fremont, CA, USA) using purified promoter DNA and digested vector in a 1 : 2 molar ratio. 2.5 µL cloning reaction was used for transformation of One Shot TOP10 chemically competent Escherichia coli (Thermo Fisher Scientific). DNA from colonies was isolated and Sanger‐sequenced (Beckman Coulter Genomics, High Wycombe, UK). For the construction of the pGL4.10‐IL33‐promoter+enhancer plasmid, the pGL4.10‐IL33‐promoter plasmid was digested with SalI, gel‐purified, and cloned with enhancer insert in a 1 : 2 molar ratio using the In‐Fusion cloning kit.

To restore expression of functional IL10RB gene in the IL-10RB^−/− patient iPSCs, we used the TALEN-mediated gene integration approach to integrate a functional copy of the IL10RB gene into the genome of the IL-10RB^−/− mutant patient iPSCs. In brief, we generated the AAVS1 EF1a-IL10RB-PGK-puro targeting vector by Gibson assembly. We transformed the Gibson assembly product into OneShot TOP10 chemically competent Escherichia coli (Thermo Fisher Scientific) and picked positive colonies. We isolated plasmids from the positive colonies and confirmed the presence and sequence of EF1α-IL10RB in the targeting vector by restriction digests, PCR and sequencing. Subsequently, the targeting vector was transformed into competent E. coli to isolate endotoxin-free plasmids to transform into the IL-10RB^−/− patient iPSCs. We transfected the mutant human iPSCs with TALEN-L (5′-CCC​CTC​CAC​CCC​ACA​GT-3′), TALEN-R (5′-TTT​CTG​TCA​CCA​ATC​CT-3′) and targeting vector via nucleofection (Amaxa Biosystems). The resultant targeted cells were selected on puromycin for 7 d. Surviving colonies were picked and expanded. The positive clones were confirmed by PCR and sequencing. All iPSC lines used in this study are karyotypically normal.

The recipient vector pCW22 (kindly shared by J. Lingner, EPFL [31 (link)]) was digested with Sal‐I and Sbf‐I to remove the Cas9 gene (4 kb) from the trAT (Tet‐On)‐containing plasmid (9.6 kb). The donor vector pEGFP‐C2 that encodes the canonical isoform 1 of HTATIP2 (CC3, UniProtKB/Swiss‐Prot, Q9BUP3‐1) [11 (link)] was kindly provided by H. Xiao, University of Michigan. The plasmid was digested by EcoRI and BamHI to isolate the insert, encoding the green fluorescent protein (GFP, 717 bp) fused with HTATIP2 (744 bp), and was gel purified (QIAquick® Gel purification kit; Qiagen, Hilden, Germany). The recipient vector and the isolated HTATIP2/GFP sequence were digested with Bam‐II and Sal‐I to generate compatible ends, and the fragments were ligated using T4 DNA ligase (Promega, Madison, WI, USA) (100 mg total DNA/reaction, ratio 1 : 3). Ligation products were transformed into One Shot® TOP10 Chemically Competent Escherichia coli (C404010; Thermo Fisher Scientific) using the protocol from the manufacturer. Surviving bacterial colonies were tested with polymerase chain reaction (PCR) for the presence of the HTATIP2/GFP‐insert, followed by sequence verification (primer sequences, Table S1). The pCW22 vector containing the HTATIP2/GFP‐inducible system was produced in E. coli and purified using the QIA Plasmid Miniprep Kit (12163; Qiagen) for lentiviral production.