7 × 10-6 for the NCIMB 11163 strain, ca. 8 × 10-8 for CU1 Rif2 and ca. 15 × 10-6 for ATCC 29191 (reported as Cm-resistant colony forming units/total colony forming units surviving electroporation). find more plasmid pZ7C was stably maintained for more than 150 generations in all three strains when cells were cultured in RM medium containing 100 μg/ml chloramphenicol (data not shown). An agarose gel of (HindIII-digested) plasmid DNA present in the three wild type (WT) and pZ7C-transformed strains is shown in Additional file 4 (Panels A, B and C: compare

the lanes marked ‘WT’ and ‘pZ7C + Cm’, respectively). The introduction Selleck CHIR98014 of pZ7C appeared to have little effect on the respective levels of the endogenous plasmids within Lenvatinib in vivo the ATCC 29191 and CU1 Rif2 strains. However, when the recombinant NCIMB 11163/pZ7C strain was propagated in RM medium containing chloramphenicol, the intensity of the band corresponding to the endogenous pZMO7 plasmid decreased markedly compared to the wild type strain (Additional file 4, Panel A). This finding indicates that there is most probably direct competition for replication between the endogenous pZMO7 plasmid and the pZ7C shuttle vector within the same cell. However, the introduction

of pZ7C had no apparent effects on the levels of the smaller endogenous pZMO1A plasmid, suggesting that it utilized a non-competing mode of replication. Equivalent results were obtained with the pZ7-184 plasmid (data not shown). Qualitative evaluation of pZ7C plasmid stability under non-selective culture conditions The stability of pZ7C within the NCIMB 11163, CU1 Rif2 and ATCC 29191 strains during propagation under non-selective conditions was investigated using a previously described approach [41]. As may be seen in Additional file 4, the levels of the pZ7C plasmid remained relatively constant within the CU1 Rif2 and ATCC 29191 strains

during this process of serial sub-culturing under non-selective conditions. This indicated that a selectable marker was not essentially required for stable maintenance of Fenbendazole the pZ7C plasmid for a period of ca. 50-70 generations in the ATCC 29191 and CU1 Rif2 strains. The situation was markedly different in the NCIMB 11163 strain, where pZ7C levels dropped to barely detectable amounts only 24 hours (10-14 generations) after the removal of the selectable marker (Additional file 4, Panel A). This was further verified by results from quantitative PCR (qPCR) experiments performed under analogous conditions (see below). Copy number determination for native pZMO1A and pZMO7 plasmids in Z. mobilis NCIMB 11163 Before performing a more detailed analysis of their plasmid copy numbers (PCN), we first determined the relative proportions of the endogenous pZMO1A and pZMO7 (pZA1003) plasmids present within Z. mobilis NCIMB 11163 using a gel-based approach.