Escherichia coli top10f

Escherichia coli TOP10F′ (Invitrogen, USA) used for cloning was grown in Luria-Bertani broth (LB) (Difco, MI, USA). R. erythropolis strain L88, a lysozyme sensible mutant was grown to 26 °C in the same culture medium used for E coli. This bacterium was used as a host for recombinants thiostrepton induced (pTip-QC1) and constitutive (pNit-QC1) plasmids [26 (link)–28 (link)].

Escherichia coli TOP10F’ (Invitrogen, Waltham, MA, USA) was used as a host for vector construction and DNA manipulation. E. coli BL21(DE3) (Invitrogen, Waltham, MA, USA) was used as a host for protein expression. Pseudomonas aeruginosa PA14 (serotype O19) was kindly provided by Dr. Hwan-You Chang (Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan). The vector pET-23a (+) (Novagen, Burlington, MA, USA) with a T7 promoter was used for recombinant protein expression in E. coli cells and sequence analysis. Takara’s Chaperone Plasmid Set (#3340, TaKaRa, Shiga, Japan) was used for chaperone co-expression. All other buffers and reagents were of the highest commercial purity.

The following strains were used in this study: Escherichia coli TOP10F′ (Invitrogen, Waltham, MA, USA), E. coli ER2566 (New England BioLabs, Ipswich, MA, USA), E. coli ER2929 Dam⁻ strain lysogenized with DE3 element [19 (link)] and Sinorhizobium sp. LM21 [16 (link)]. Sinorhizobium sp. LM21 was grown in tryptone-yeast extract (TY) medium [20 ] at 30 °C. E. coli strains were cultured under standard conditions in lysogeny broth (LB) medium at 37 °C. When required, media were supplemented with kanamycin (Km) at 50 μg mL⁻¹. Plasmids pET28a and pET30a (Invitrogen, Waltham, MA, USA) were used as expression vectors.

The plasmid encoding the ApN gene (p-ApN) was constructed by inserting the mouse full-length ApN cDNA45 (link) into the pcDNA™3.1D/V5-His-TOPO^® vector (pcDNA™3.1 Directional TOPO^® Expression Kit from Invitrogen, Thermo Fisher Scientific, Gent, Belgium). An empty plasmid was used as control. The plasmid containing the precursor miRNA for Mus musculus miR-711 stem-loop with Enhanced Green Fluorescent Protein (EGFP) reporter gene (p-miR-711) as well as its negative control were purchased (GeneCopoeia, Rockville, USA). All plasmids were amplified in Escherichia coli top 10F’ (Invitrogen), purified with an EndoFree plasmid giga kit (Qiagen, Venlo, The Netherlands), and then were stocked at −80 °C.

All chemicals used in the study were analytical grade and bought from Sigma (Saint Louis, USA) or Chempur (Piekary Śląskie, Poland). The brown coal used in the study was obtained from Bełchatów Brown Coal Mine (Poland). Coal particles with a diameter of 1–2 mm were subjected to pretreatment with 8M HNO₃ as described earlier (Romanowska et al. 2015 (link)) and then subjected to microbial solubilization.
The microorganism used in the study was F. oxysporum LOCK 1134 (Centre of Industrial Microorganisms Collection—LOCK, WDCM105), strain isolated from brown coal from Bełchatów Brown Coal Mine, currently in pure culture collection at the Institute of Technical Biochemistry, Lodz University of Technology. Pichia pastoris KM71H from Invitrogen (Carlsbad, USA) was used for protein expression and Escherichia coli top10f’ (Invitrogen, Carlsbad, USA) for standard cloning procedures. pJET1.2 plasmid (Thermo Fisher Scientific, Walthman, USA) was used for basic molecular manipulations and pPICZαA vector (Invitrogen, Carlsbad, USA) was used for enzyme expression.

The plasmid encoding the ApN gene (p-ApN) was constructed by inserting the mouse full-length ApN cDNA [33 (link)] into the pcDNA™3.1D/V5-His-TOPO® vector (pcDNA™3.1 Directional TOPO® Expression Kit from Invitrogen, Thermo Fisher Scientific, Gent, Belgium) [11 (link)]. An empty plasmid was used as the control. The plasmid containing the precursor miRNA for Mus musculus miR-711 stem-loop with enhanced green fluorescent protein (EGFP) reporter gene (p-miR-711) as well as its negative control were purchased (GeneCopoeia, Rockville, MD, USA). All plasmids were amplified in Escherichia coli top 10 F′ (Invitrogen), purified with an EndoFree Plasmid Giga Kit (Qiagen, Venlo, The Netherlands) and then stocked at −80 °C.

The LDK coding sequence was cloned into the pIR1_SAT vector to generate the add-back parasites. After ligation, the resulting plasmids were incubated with Escherichia coli TOP10F’ (Invitrogen, Waltham, MA, USA) at 4°C for 30 minutes and then at 42°C for 45 seconds using a dry bath. Following this, the bacteria were incubated at 37°C for 1 hour to induce expression of the ampicillin resistance gene and subsequently plated on solid LB medium containing ampicillin (Sigma, St. Louis, MO, USA). Clones possessing the correct LDK sequence, confirmed via colony PCR, were subjected to digestion with the restriction enzymes BglII (BioLabs, Durham, NC, USA), SacII (Promega, Madison, WI, USA), and EcoRV (Promega) to verify the orientation of the gene within the plasmid. Subsequently, 100 μg of the pIR1_SAT_LDK plasmid was linearized with using the restriction enzyme SwaI (BioLabs) and precipitated using sodium acetate and isopropanol. Transfection of ΔLDK mutant promastigotes with the pIR1_SAT_LDK plasmid was conducted following the established protocol [16 (link)], and the parasites were selected using 100 μg/mL of nourseothricin sulfate (Sigma).