Nucleospin plasmid kit

Four different Myc-tagged DARPins targeting Lamin A/C (LaA_1, LaA_2, LaA_3 and LaA_4) and control myc-tagged DARPin (E3.5) were generously provided by Ohad Medalia [23 (link)]. Coding sequences were received in pEGFP-N1 plasmid where EGFP was replaced by IRES-GFP (pEGFP-DARPin-IRES-GFP plasmids). Subcloning of the different myc-tagged DARPins into an HIV-derived vector plasmid (pRRL-SIN-cPPT-DesminGFP-HYGRO-WPRE, hereafter pRRL-EGFP) plasmid was achieved by digestion of pEGFP-DARPin-IRES-GFP plasmids by Nhe I/Pml I and ligation in place of EGFP within the XbaI/Pml I sites of pRRL-EGFP, and grown in STBL2 bacteria (ThermoFischer Scientific, Illkirch, France) at 32 °C. Plasmid purifications was performed using NucleoSpin Plasmid kits from Macherey Nagel. The integrity of plasmid sequences was validated by sequencing. Stocks of vesicular stomatitis virus GP pseudotyped self-inactivating lenti-viral vectors were produced in 293 T cells using a four-plasmid system as described previously [24 (link)]. Supernatant was collected during 4 days and added to mouse primary myoblast culture medium at a 1:2 and 1:4 ratio. Three days post-infection, cells were split for further amplification with proliferation medium containing 50 µg/mL Hygromycin (Thermofisher scientific, illkirch, France) for 3 weeks for the specific selection of DARPin positive myoblasts.

All chemicals were either from Sigma or Roth. Tetracycline-hydrochloride (#T7660) was from Sigma-Aldrich, tigecycline was from Pfizer (New York City, NY, USA), and omadacycline was supplied by Paratek Pharmaceuticals (Boston, MA, USA). Plasmid DNA was isolated using NucleoSpin Plasmid kits (Macherey & Nagel). DNA oligonucleotide primers were from Eurofins Genomics and [γ-³²P]-ATP was from PerkinElmer.

Potassium hydroxide pellets, acetone (≥98%), ergosterol (≥98%) and squalene (≥98%) used as standards and petroleum ether (b.p. = 40–60°C) were all purchased from Sigma-Aldrich, while ethanol absolute was supplied from Scharlau.; Sclareol, kindly provided by VIORYL, SA, (−)-trans-caryophyllene (Sigma, C9653-5), were used as standard compounds; MyTaq DNA polymerase (BIO-21105, Bioline), and Accuzyme DNA polymerase (BIO-21051, Bioline) were used in PCR amplifications; NucleoSpin Plasmid Kit (REF 740588.250, Macherey-Nagel) was used for plasmid DNA purification; QIAquick Gel Extraction Kit (#28704, Qiagen) was used for gel extraction and DNA purification.
Yeast media: D (+)-Glucose monohydrate (16301, Sigma); Yeast Nitrogen Base w/o AA, carbohydrate & w/AS (Y2025, US Biologicals); Complete Minimal (CM) medium is composed of 0.13% (w/v) dropout powder (all essential amino acids) [45 ], 0.67% (w/v) yeast nitrogen base w/o AA, 2% glucose; TOPO TA Cloning Kit Dual Promoter (K4610-20, Invitrogen). All yeast transformations were done by lithium acetate transformation.

The cDNAs encoding mutant p105 and p50 were purchased from GeneArt Gene Synthesis (Invitrogen) or generated by site-directed mutagenesis and subcloned into the pEGFP-C1 expression vector (Takara/Clontech) to generate N-terminally EGFP-tagged constructs. Competent E.coli (NEB, 5-alpha) were transformed with the vectors constructs and plasmids were isolated with NucleoSpin Plasmid Kit (Macherey-Nagel).

The positive clone (pCOMB3x-VHH_19) was first purified with the NucleoSpin^® Plasmid Kit (Macherey-Nagel, Dueren, Germany) and sequenced by LGC Genomics GmbH (Berlin, Germany) with M13rev2 sequencing primer provided by the company. The resulting DNA sequence was translated into an amino acid sequence (Kabat numbering scheme) and analyzed for the identification of the four hallmarks indicating a camelid single-domain antibody (VHH). pCOMB3x-VHH_19 was transferred into chemically competent non-suppressor E. coli strain HB2151 and expressed as previously described in [44 (link)]. The soluble His-tagged VHH_19 was purified from the periplasmic fraction via Ni-NTA affinity chromatography according to the manufacturer’s standard protocol (Protino^® Ni-NTA Agarose, Macherey-Nagel, Dueren, Germany).

For YFP-labeling of A. radicis N35 araI::tet, plasmid pBBR1MCS-2-YFP, a YFP expressing broad-host range vector, and for GFP-labeling of the N35 wild type, plasmid pBBR1MCS-2-GFP were used. After isolation using a NucleoSpin plasmid kit (Macherey & Nagel, Düren, Germany) the plasmid was transferred to electro-competent cells of A. radicis N35 as described by Dower et al. (1988 (link)). Electroporation was performed with a Gene Pulser instrument (Bio-Rad, Munich, Germany) using a voltage of 2.5 kV for 4.5–5.5 ms. The resulting transformants were selected on Km containing NB plates and examined for specific fluorescence with an epifluorescence microscope at an excitation wavelength of 488 nm.