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	Protoplast of S. lividans ΔTA-pKC796-Tox(pGM160-YefMsl) and the control strains, S. lividans wt-pKC796 and S. lividans ΔTA-pKC796(pGM160-YefMsl) were transformed with the expression plasmids. After transformation, protoplasts were regenerated o/n at 28°C, then overlayed with 50 μg/mL neomycin to select the cells transformed with the expression plasmids and transferred to 37°C for 3 days to eliminate the temperature-sensitive plasmid (pGM160-YefMsl).

Protocol tips

Protoplast of S. lividans ΔTA-pKC796-Tox(pGM160-YefMsl) and the control strains, S. lividans wt-pKC796 and S. lividans ΔTA-pKC796(pGM160-YefMsl) were transformed with the expression plasmids. After transformation, protoplasts were regenerated o/n at 28°C, then overlayed with 50 μg/mL neomycin to select the cells transformed with the expression plasmids and transferred to 37°C for 3 days to eliminate the temperature-sensitive plasmid (pGM160-YefMsl).

Publication protocol

Protoplast of S. lividans ΔTA-pKC796-Tox(pGM160-YefMsl) and the control strains, S. lividans wt-pKC796 and S. lividans ΔTA-pKC796(pGM160-YefMsl) were transformed with the expression plasmids. After transformation, protoplasts were regenerated o/n at 28°C, then overlayed with 50 μg/mL neomycin to select the cells transformed with the expression plasmids and transferred to 37°C for 3 days to eliminate the temperature-sensitive plasmid (pGM160-YefMsl). The loss of the temperature-sensitive plasmid (pGM160-YefMsl) in these transformants colonies was checked by replica-plating the clones onto R2YE plates with 10 μg/mL thiostrepton. Finally, the clones were reinoculated on patches on R2YE plates without antibiotics and incubated at 28°C.

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