Quantitative real time PCR (qPCR) was used for a more accurate determination of the respective plasmid copy numbers, according to the method described by Skulj et al.. Using this relative quantification approach, the PCN is determined by quantifying the number of plasmid molecules per chromosome molecules in each sample using specific qPCR primer sets. We
designed two sets of qPCR primers for each plasmid, which targeted distinct loci: the rep and mob genes of pZMO7, as well as click here the rep gene and a non-coding region of the pZMO1A plasmid (see Additional file 1). The polyphosphate kinase 2 (ppk2) gene, a highly-conserved single copy gene present on the chromosomes of all characterized Z. mobilis strains [ATCC 29291: ZZ6_0566; NCIMB 11163: Za10_0556; ATCC10988 (CU1 Rif2 parent): Zmob_0569] was LEE011 ic50 selected as a reference genetic locus for the determination
of Z. mobilis chromosome copy number. The two respective pairs of qPCR primers that targeted distinct regions on the pZMO1A or pZMO7 plasmids were then directly compared, to investigate whether or not there were notable differences in the PCN values obtained. The PCN for pZMO7 was determined to be 1.2 ± 0.1 when the rep gene was targeted, and was 1.4 ± 0.1 when the mob gene was targeted. In analogous experiments, the PCN of pZMO1A was found to be 5.0 ± 0.2 using the primer pair that targeted the rep gene, and was 5.3 ± 0.4 using the primer pair that targeted a predicted non-coding region of the plasmid. This data correlated closely with the estimates of relative AZD1080 supplier pZMO1A and pZMO7 plasmid abundances determined using gel-densitometry (see above). The consistent nature of the PCN values obtained indicated that both of the respective pairs of qPCR primers had equivalent target specificities
and amplification efficiencies. We next used qPCR to investigate whether the PCNs of pZMO7 and pZMO1A in cultured Z. mobilis NCIMB 11163 cells varied considerably during the different phases of growth (Additional file 5). It was found that PCN of of pZMO7 was relatively consistent throughout the growth phases, fluctuating slightly at around 1.2 copies per chromosome. The PCN of pZMO1A was around 4.5 to 5 during the lag and exponential phases, declining to around 3.0 during the stationary phase. Copy number determination for pZMO7-derived shuttle vectors in the Z. mobilis NCIMB 11163, ATCC 29191 and CU1 Rif2 strains A similar qPCR strategy was employed to investigate the copy numbers of the pZMO7-derived pZ7C and pZ7-184 plasmids, which had been established within the Z. mobilis NCIMB 11163, ATCC 29191 and CU1 Rif2 strains. We designed and utilized a qPCR primer pair targeting the chloramphenicol acetyl transferase (cat) gene; so that the PCNs of pZ7C and pZ7-184 could be distinguished from those of the native pZMO7 plasmids within the NCIMB 11163 strain (Additional file 1). This enabled PCNs to be directly compared between the three strains. Results are summarized in Table 2.