Elepo21

S. pombe cells were transformed by electroporation using an ELEPO21 (Nepa Gene, Chiba, Japan). The cells were then grown to 2 × 10⁷ cells/mL, placed on ice for 15 min, and collected by centrifugation. The cell pellet was suspended in 0.1 M lithium acetate and incubated for 45 min at the appropriate culture temperature (i.e., 30 °C for HM123, YTP1847, YTP1889 cells and 26 °C for YTP1840, YTP1901 cells or 23 °C for Sp525 cells). Next, 1 M DTT was added to a final concentration of 10 mM, and the suspension was incubated for 15 min at the appropriate culture temperature. After centrifugation, the cell pellet was washed twice with ice-cold 1 M sorbitol. The final pellet was resuspended in ice-cold 1 M sorbitol at a volume that results in a solution containing 1 × 10⁹ cells/mL. The cell suspension was mixed with 10 ng plasmid and then transferred to an ice-chilled cuvette with a 2 mm gap. The electroporation conditions were as follows: poring pulse—1500 V, pulse length 3.5 ms, pulse interval 50 ms, two pulses, polarity +; transfer pulse—100 V, pulse length 50 ms, pulse interval 50 ms, three pulses, polarity +/−. The resistance value was adjusted to approximately 45–20 kΩ just before electroporation. After electroporation, the cells were immediately added to ice-cold 1 M sorbitol and plated on selection medium.

The reverse transcription reaction was conducted using SuperScript IV Reverse Transcriptase and oligo (dT)₂₀ primer. A first PCR was performed using primeSTAR DNA polymerase (Takara Bio), first strand cDNA, and VNAR-specific primers. The resultant PCR mixtures were diluted 100-fold with water, and the diluted product was applied to the nested PCR. To create pYES3-VNAR, the Aga2 signal sequence, NheI and BamHI sites, AGIA tag, and Aga2 mature peptide fusion sequence were inserted into the pYES3 vector (Thermo Fisher Scientific). NheI- and BamHI-digested pYES3-VNAR was mixed with purified and nested PCR products, and the mixture was transformed into electrically competent EBY100 yeast (ATCC) (Chao et al., 2006 (link)) using ELEPO21 (Nepa Gene). Four transformations and the dilution plates demonstrated that 1.5 × 10⁷ total transformants were generated for the VNAR library. The yeast was cultured according to previous reports (Chao et al., 2006 (link)).

The assembled genomes (10 μl) were electroporated into E. coli HST08 Premium Electro-Cells (Takara Bio Inc., Shiga, Japan) with ELEPO21 (Nepa Gene Co., Ltd., Chiba, Japan; poring pulse [voltage: 1500 V, pulse length: 2.5 ms, pulse interval: 50 ms, number of pulses: 1, polarity: +], transfer pulse [voltage: 150 V, pulse length: 50 ms, pulse interval: 50 ms, number of pulses: 5, polarity: +/−]). After electroporation, samples were added to 1 ml SOC medium (Takara Bio Inc.) and incubated at 37 °C for 20 to 60 min with shaking at 200 rpm. The samples (500 μl to 1 ml) and E. coli O157 strain ATCC 43888 overnight cultures (200–300 μl) were added to 3 ml of LTA. They were poured onto LB plates and incubated at 37 °C overnight. The plaques were counted, and synthetic phages were kept in 100 μl of SM buffer at 4 °C for the following experiments.