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.