Figure 2

Selection of the bacterial excisants with the araC-Cre insert in the chromosomal lacZ gene. (A) The passaged bacterial population, which contained the excisants, was plated at 44°C to obtain individual colonies and to encourage the loss of the excised plasmids. Screening revealed 10 partially Cm-sensitive clones, which were proven to be mixtures of Cm-sensitive (plasmidless) and Cm-resistant (plasmid-bearing) cells. Subcloning resulted in isolation of completely Cm-sensitive plasmidless clones. (B) The obtained Cm-sensitive subclones were screened for the presence of the Cre gene by PCR using primers CRF and CRR homologous to the Cre gene. Bacteria were added directly to the PCR reaction mixtures. (C) Finally, the unwanted plasmid pGETrec was expelled from the Cre-positive clones by the incompatible minicircle producer plasmid pFIXluc maintained by selection for Cm-resistance.