However, transformation efficiencies for mycelial fungi are highly restricted by difficulties in colony formation and competent cell preparation.
Standard procedures for plasmid isolation, restriction enzyme digestion, ligation, competent cell preparation and transformation were used as described by (Sambrook et al., (1989)).
All E. coli strains were cultured in Luria Bertani (LB) medium (Sambrook and Russell 2001) or on LB agar plates containing 100 mg/l ampicillin except that for competent cell preparation.
While in electroporation of obligate Gram-positive anaerobes such as some thermophilic Clostridia, custom-made electroporators that are until now not accessible to most laboratories are required [1], [3], and a large anaerobic workstation for competent cells preparation and applying electro-pulses has to be equipped.
The Competent Cell Preparation Kit was from TaKaRa (Dalian, China).
Plasmid methylation with MspI methyltransferase, competent cell preparation, and electroporation of C. cellulolyticum were precisely performed as described in previous studies [ 11, 14].
RNA extraction, competent cell preparation, plasmid DNA isolation, ligation, and transformation in this study were performed according to standard molecular cloning protocol.
E. coli JM109 competent cell preparation and heat-shock transformation were carried out according to standard protocols (Sambrook and Russell 2001).
The overnight culture were then inoculated into 50 ml LB medium and grown at 37°C for 3 h by which time the optical density of the culture had reach 0.5 of OD600 and could be used for competent cell preparation and protein expression.
To do so, we prepared column-depleted perforin-competent SCILs preparations deficient in CD8+ cells, CD4+ cells, or NK1.1+ cells.
No plasmid-like bands were detected by agarose gel electrophoresis, and transformation of E. coli ultra-competent cell preparations and selection on kanamycin plates was also unsuccessful.
