Fig. 1

Optimization of the conditions for gene transfer by electroporation in T. asahii. A Structure of the DNA fragment for constructing the cnb1 gene-deficient mutant and the predicted genome of the cnb1 gene-deficient mutant. B Scheme for obtaining drug-resistant strains by gene transfer via electroporation. C Effect of the number of incubation days for preparing competent T. asahii cells. The T. asahii MPU129 ku70 gene-deficient mutant was spread on SDA and incubated at 27 °C for 1, 2, or 5 days. The PCR-amplified 5'-UTR (cnb1) -NAT1-3'-UTR (cnb1) fragment (180 ng/2 µl) was added to the competent T. asahii cells (40 µl) and electroporated (time constant protocol: 1.8 kV, 5 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted. D Effect of voltage on gene transfer by electroporation. The PCR-amplified 5'-UTR (cnb1) -NAT1-3'-UTR (cnb1) fragment (180 ng/2 µl) was added to competent T. asahii cells (40 µl) prepared by culture for 1 day and electroporated (time constant protocol: 1.2–2.1 kV, 5 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted. E Effect of time constant on gene transfer by electroporation. The PCR-amplified 5'-UTR (cnb1) -NAT1-3'-UTR (cnb1) fragment (180 ng/2 µl) was added to competent T. asahii cells (40 µl) prepared by culture for 1 day and electroporated (time constant protocol: 1.8 kV, 3–10 ms). The number of colonies grown on SDA containing nourseothricin (300 µg/ml) was counted