2.6 The subculture of strains and plasmid stability examination
Strain YZ72-5, harboring plasmid for IUP expression with chloramphenicol resistance, was cultivated in LB liquid media containing 25 μg/mL CmR in 37℃ for 12 h. Cells were sub-cultured for three generations, after each cultivation in LB liquid medium with 25 mg/mL CmR under 37 ℃ for 12 h, strains were scribed to the CmR-free LB solid plate. Then, strains were cultivated 37 ℃ for 12 h, obtaining single colonies. Afterwards, 100 single colonies were selected to grow on CmR-containing LB solid plate and CmR-free LB solid plate. After cultivation for 12 h at 37 °C, colonies growing on LB solid plate but not on LB with 25 μg/mL CmR were interpreted that the plasmid has been lost. Each experiment was carried out in three parallel groups. The plasmid stability was calculated as the number of samples with growth in LB with antibiotics/100× 100%.
Strain YZJ3-4 was plated on LB solid plate with 100 mg/mL AmpR, which was then cultivated in 37 ℃ for 12 h, obtaining single colonies. Afterwards, strains on the plate were washed using fresh LB liquid medium, then they were subscribed to plates with 100 mg/mL AmpR, which procedure was circulated. The times of “generation” means the times the process was carried out.