The procedure is described as follows. Cells were grown in ASSPL to early log phase (OD600 nm of 0.15–0.2) and harvested by centrifugation. Harvested cells were washed twice with cold electroporation buffer (10% glycerol+1 mM MgCl2) and centrifuged at 4000 g for 10 min at 4 °C. The cell pellet was resuspended in the same electroporation buffer to 1/50 volume of the original culture. Eighty microliters of this cell suspension was mixed with 2 μg of genomic DNA or PCR amplicon on ice and transferred to a precooled 0.1-cm gap electroporation cuvette (BTX, Harvard Apparatus). The cell–DNA mixture was subjected to electroporation
at field GSK-3 beta pathway strength of 20 kV cm−1, capacitance of 25 μF, and resistance of 200 Ω. Following electroporation, the cells were immediately diluted in 1 mL of THL medium and incubated anaerobically for 16 h at 37 °C then plated on THL agar plates
supplemented with 1 mg mL−1 streptomycin. Colonies would appear after 24–48 h. Using this protocol, we were able to consistently obtain 9–12 colonies μg−1 mutant genomic DNA, which was two to three times higher than the number of colonies from the wild-type DNA (3–5 colonies μg−1 DNA and these colonies are spontaneous mutants). This result suggested that at least half of the streptomycin-resistant colonies obtained using the mutant DNA contained introduced mutations while the rest may have originated from spontaneous mutation. We could not obtain consistent transformation results when using other parameter combinations mentioned in Materials and methods. Volasertib in vitro With the optimized protocol, we next tested whether PCR-generated DNA could be used to transform V. parvula PK1910. PCR amplicons were generated with the primers rpsLup-F and rpsLdn-R (Table 2 and Fig. 1) using the wild-type and the spontaneous streptomycin-resistant strains SR1 (AAG to AAC mutation) and SR2 (AAG to AAT mutation) as templates. The amplicons were named rpsL-WT, rpsL-SR1, and rpsL-SR2, respectively (Fig. 1). The three PCR amplicons were transformed into PK1910 Sclareol with the procedure described above. In five
separate experiments, we obtained similar results as the transformation with genomic DNA: there were always about two times more colonies in the transformation with the mutant DNA than with the wild-type DNA. For one of these experiments, we sequenced the rpsL gene of all the colonies that appeared on the plates. As shown in Table 3, most colonies in the rpsL-SR1 transformation have AAC mutation in codon 43, while most colonies in the rpsL-SR2 transformation have AAT mutation in codon 43. The colonies in rpsL-WT transformation, representing the spontaneous mutation, have a similar distribution of the AAC or AAT mutation in codon 43. This result strongly suggests that DNA-mediated transformation had occurred in V.