Micropulser

For preparation of electrocompetent cells, bacteria were grown to OD₆₀₀ = 0.5–0.6, and pelleted by centrifugation at 4°C. Two series of washes and centrifugations (6000 rpm) of 1 vol milliQ water and a final wash in 1/50 vol 10% glycerol at 4°C were applied. Cells were resuspended in 1/500 vol 10% glycerol and aliquotted in 50 μl samples. K56-2 electrocompetent cells were transformed with <10 ng of the pTnMob-OCm plasposon DNA (Dennis and Zylstra, 1998 (link)) in a 0.2 cm Gene Pulser^® cuvette (BioRad) and subjected to an electric pulse (2.5 kV, 25 Mf, and 200 Ω) in a MicroPulser TM (BioRad). Electroporated cells were added to 1 ml LB and incubated with shaking at 37°C to allow expression of antibiotic resistance genes. After incubation cells were plated on antibiotic containing media. The pool of bacterial colonies growing on Cm plates was used as donor cells to test Ptw conjugation. Considering the size of the plasmid and the total length of the genomic DNA, it is estimated that on average one out of 100 colonies would have the plasposon inserted in the Ptw plasmid, and from those, about half would not affect any transfer-related functions. Therefore, in a mating experiment involving 10⁶ donor cells, we would expected to have at least 5 × 10³ cells carrying a Cm-resistant, transfer-proficient Ptw plasmid.

The PG-1 DNA fragments, PG-1-ATG-For and PG-1-TAC-Rev, (Table S3) were annealed and purified using phenol chloroform (Sigma Aldrich). The annealed DNA fragment contained ATG and TAG overhangs were ligated into the AlwNI digested pET31b vector (Novagen) downstream of the KSI gene (KSI-PG1) and transformed into E. coli BL21 (DE3) (Invitrogen, CA, USA). When the culture reached an OD₆₀₀ of 0.6 in LB medium, the expression of the insert was induced with 0.1 mM IPTG (Sigma Aldrich). The pET31b construct carrying KSI was expressed in the same way. At various time intervals, cell viability was estimated and proteins were extracted and separated in SDS-PAGE.
PG-1 was PCR amplified using the primer set (pPIC-BamHI-PG1-For and PG1-EcoRI-pPIC-Rev, Table S3) using pig cDNA. The amplified PG-1 fragment and pPIC3.5 vector (Life Technologies, Seoul, South Korea) were digested with BamHI and EcoRI (NEB), ligated, and transformed. The plasmid pPIC3.5 containing PG-1 was linearized using SacI (NEB) and integrated into the P. pastoris GS115 (Life Technologies) genome using an electroporator (MicroPulser^TM, Bio-rad, CA, USA). Protein expression was carried out using the Pichia Expression Kit (Life Technologies) in buffered and minimal methanol medium according to the manufacturer’s protocol.

For preparation of electrocompetent cells, bacteria were grown to OD600 = 0.5–0.6, and pelleted by centrifugation at 4°C. Two series of washes and centrifugations (6,000 rpm on a Beckman JA-10 rotor) of 1vol milliQ water and a final wash in 1/50 volume 10% glycerol at 4°C were applied. Cells were resuspended in 1/500 vol 10% glycerol and aliquoted in 50 μl samples. Aliquots were frozen on dry ice and kept at −70°C until usage. Aliquots were mixed with < 10 ng of DNA in a 0.2 cm Gene Pulser^® cuvette (BioRad) and subjected to an electric pulse (2.5 kV, 25 μF and 200 Ω) in a MicroPulser^TM (BioRad). Electroporated cells were added to 1 ml LB and incubated with shaking at 37°C to allow antibiotic expression. After incubation cells were plated on antibiotic containing media.

The PCR products were cloned using TOPO Zero Blunt PCR cloning kit and then subcloned into the pSV2 expression vector using HindIII and SacI. The resulting vectors were transformed into BMB171 cells via electroporation using a Bio-Rad Micropulser^TM. BMB171 cells were grown in LB-glycine 0.12% up to an OD600 of 0.15, corresponding to the early exponential phase, and then transformed with 500 ng of the constructs. The electroporation conditions were 2 kV/cm, 200 Ω, and 25 μF for 4 ms. Transformed cells were revitalized via incubation in 500 μl of LB for 2 h at 30°C at 50 rpm. Two hundred microliters of transformed cells were plated on 60-mm Petri dishes containing LB agar supplemented with 6 μg/ml chloramphenicol. Colony counts and percent efficiency of transformation were calculated after 48 h. Endospore and crystal formation was evaluated via scanning electron microscopy (SEM).

The transformation procedure was adapted from the protocol developed by De Backer et al. (1999 (link)) for the transformation of C. albicans by electroporation. In short, an overnight culture of C. intermedia CBS 141442 grown at 30°C and 1 × g shaking was used to start a culture at OD₆₀₀ = 0.1 in 100 ml YPD (1% yeast extract, 2% bacto peptone, and 2% glucose). Once cells reached OD₆₀₀ = 1, the culture was centrifuged (2600 × g for 3 min) and washed twice with ice-cold sterile deionized water and once with ice-cold 1 M sorbitol. Cells were centrifuged once again followed by resuspension in 160 μl of 1 M sorbitol. The resuspended cells were divided into 40 μl aliquots in microcentrifuge tubes. To each aliquot, 8 μl of DNA (with an optimal amount of 2 μg) was added. The transformation mix was transferred to a chilled electroporation cuvette (0.2 cm gap BioRad MicroPulser electroporation cuvettes, BioRad Laboratories, Hercules, CA, USA) and electroporated with a BioRad MicroPulser (BioRad Laboratories) at 1.5 kV. Cells were thereafter gently transferred to 1 ml of 1 M sorbitol in YPD and incubated at 30°C for 3 h with 1 × g of shaking. Cultures were spread on YPD agar (2%) plates with cloNAT (200 μg/ml) for selection and left to grow at 30°C for 48 h or until colonies emerged.