Biorad gene pulser 2

pSFV containing respective transgenes and a SFV-Helper2 plasmid were linearized by digestion with SpeI (Life Technologies) for RNA synthesis by SP6 polymerase (Amersham Pharmacia Biotech, Piscataway, US) mediated in vitro transcription in the presence of capping analog (Life Technologies). Subsequently, pVREP RNA and SFV-Helper-2 RNA were mixed in a 2:1 ratio and co-transfected in BHK-21 cells in the presence of electroporation buffer using the BioRad Gene Pulser II (2 pulses of 850 V / 25 μF; BioRad, Hercules, US). After electroporation, the cells were cultured in RPMI-1640 medium supplemented with 5% (v/v) fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin for 48 hours at 30°C with 5% CO₂. The rSFV particles were purified by means of discontinuous sucrose density gradient ultracentrifugation and stored in TNE buffer as aliquots at -80°C. Before use, all rSFV particles were activated by the addition of 1:20 volume 10 mg/ml α-chymotrypsin (Sigma Chemical, St. Louis, US) and 2 mM CaCl₂ for 30 minutes to cleave the mutated spike proteins. After which, the α-chymotrypsin was inactivated by the addition of 1:2 volume 2 mg/ml aprotinin (Sigma Chemical).

JEV VLPs were produced with the pVAX-JEi plasmid derived from the pCBJE plasmid [50 (link)], which encodes the prM and E protein regions of the SA14 strain genome. This plasmid was also used as the template for introducing mutations into the E protein by use of a site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described [50 (link),51 (link)]. The pVAX-JEi 101/106/107, 306, 329, 331, 332 and 389 amino acid mutants were introduced by mutagenesis primers (S1 Table), according to the manufacturer’s protocols, and mutation was confirmed by sequencing. The JEV VLP-expressing plasmids were electroporated into COS-1 cells by use of a 0.4-cm–electrode-gap cuvette and a Bio-Rad Gene Pulser II (Bio-Rad Laboratories, Hercules, CA) at 250 V and 975 μF; electroporated cells were recovered overnight at 37°C and incubated at 28°C to enhance VLP secretion. The secreted VLPs were analyzed by Ag-ELISA and used to evaluate the presence of epitope-specific antibodies.

Sulfolobus was transformed essentially as described by Schleper and colleagues [7 (link)]. Cultures of Sulfolobus S441 (5 mL) were grown from frozen stocks for 48–72 h in a 70 °C shaking incubator. Starter cultures were transferred to 50–100 mL of fresh YS media in long neck Erlenmeyer flasks and grown until OD_{600 nm} reached ~0.20. Then, 50 mL of cells were removed and placed on ice for 30 min. Cells were washed three times with decreasing volumes of 20 mM ice-cold sucrose as described previously [7 (link)]. After the final wash, cells were resuspended in 400 μL ice-cold 20 mM sucrose and kept on ice. Then, 100 μL of cells were added to a chilled 0.1 cm gap length electroporation cuvette (Bulldog Bio, Portsmouth, NH, USA) and 2 μL of SSV or shuttle vector DNA (100–500 ng/μL) purified from E. coli using the GeneJET plasmid purification kit (Thermo-Fisher, Waltham, MA, USA) was added to the cells. Cells were transformed by electroporation (BioRad Gene Pulser II, Bio-Rad Laboratories, Hercules, CA, USA) under the following conditions: 1.5 kV, 400 Ω, 25 μF. Immediately following electroporation, cells were resuspended in 1 mL of 70 °C YS, transferred to a 1.5 mL tube, and incubated for 1 h in a 70 °C dry incubator. Following incubation, cells were transferred to 50 mL of preheated YS in a long neck Erlenmeyer flask and grown with shaking at 70 °C.

E14 cells were passaged twice and plated at 8 × 10⁴ cells/cm² for 24 h prior to transfection. Cells were transfected with a total of 2 µg DNA (i.e. pairs of Cas9-2A-EGFP vectors, each expressing single gRNAs: US1+DS1 or DS2) using the Amaxa P3 Primary cell 4D-Nucleofector X Kit (Lonza) and incubated with supplemented GMEM for 36 h. Cells were then resuspended in Dulbecco’s phosphate buffered saline (DPBS, Sigma), diluted in sterile Baxter water (TPS Healthcare/Baxter), supplemented with 2% FBS and kept at 4 °C. The GFP⁺ cells were collected with a FACS Aria IIIu cell sorter (BD). To generate single cell clones of CRISPR-modified E14 cells, sorted pools of GFP⁺ cells were plated on 0.1% gelatin-coated 100 mm culture dishes at a density of 18 cells/cm² and single colonies picked approximately 2 weeks later. RAW 264.7 cells (5 × 10⁶) were resuspended in 250 µL of culture medium containing 10 µL of DPBS ± 20 µg DNA (as pairs of FIRE-targeting Cas9-2A-EGFP vectors, as specified above) and incubated at RT for 10 min. Electroporation was performed in 0.4 cm electroporation cuvettes (BioRad) using the BioRad Gene Pulser II (BioRad), at 320 V and a capacitance of 950 µF. Cells were cultured for 36 h and GFP⁺ cells were single-cell sorted into polystyrene flat-bottom 96-well plates, using a FACS Aria IIIu cell sorter (BD).

A H. denitrificans culture (400 ml) grown in minimal medium containing 24.4 mM methanol was harvested during early exponential phase at an optical density at 600 nm (OD₆₀₀) of 0.3 (4000 × g, 10 min, 4°C). Cells were washed twice with ice-cold water (4000 × g, 10 min, 4°C), once with ice-cold 10% (v/v) glycerol and finally resuspended in 800 µl of 10% glycerol. 50 µl aliquots of cells were mixed with 500 ng of plasmid DNA and incubated on ice for 10 min. Electroporation was carried out in 0.1 cm gap cuvettes (Bio-Budget Technologies GmbH, Krefeld, Germany) with a Bio-Rad gene pulser II (Bio-Rad Laboratories) with the following electrical settings: 2.4 kV and 200 Ω at a capacitance of 25 µF. After electroporation, 1 ml of minimal medium containing 24.4 mM methanol was added to the cuvette. Cells were transferred to an Eppendorf tube and incubated at 30°C for 6 hr. Transformants were selected by plating suitable dilutions of electroporated cells onto minimal medium agar containing 24.4 mM MeOH and the appropriate antibiotics. Plates were incubated at 30°C for up to 14 days. The resulting antibiotic resistant colonies were screened via PCR.

Human CRISPR 3-plasmid activation pooled library (SAM) was a gift from Feng Zhang (Addgene #61426) [24 (link)]. SAM library amplification was carried out as described using Endura electrocompetent cells (Lucigen Corporation, Middleton, WI, USA) and BioRad Genepulser II (Bio-Rad Laboratories GmbH, Munich, Germany). DNA was prepared using Endofree Maxi or mini kits (Qiagen, Hilden, Germany) following the manufacturer’s protocol.

All of the bacterial strains and plasmids used in this study are listed in Table 1. P. protegens strains were cultured at 30°C in Luria-Bertani (LB) medium. Escherichia coli strains were routinely grown at 37°C in LB medium. P. protegens cells were electroporated using a Bio-Rad Gene Pulser II (Bio-Rad, CA, United States) at 1.8 kV and 300 Ω. Antibiotics were used at the following concentrations: kanamycin 50 μg mL^–1; ampicillin 50 μg mL^–1; streptomycin (Str) 16 μg mL^–1; tetracycline 15 μg mL^–1; gentamycin 10 μg mL^–1 (for E. coli), or 50 μg mL^–1 (for P. protegens); and chloramphenicol 34 μg mL^–1.