Figure 1.
Schematic representation of the replacement of the origin of replication of pBR322 derived vectors.
The relative position of RNAI, Rom, and the ori are depicted. When RNAI is retained in the nascent vector, vector incompatibility results upon co-transformation with pBR322-derived vectors. Removal of both RNAI and Rom yields a truly compatible vector.
Table 1.
Primers used for cloning and qPCR.
Figure 2.
Standard curve used to quantify plasmid copy number in E. coli.
Plasmid standard curves were generated using known amounts of plasmid DNA. The linear dynamic range was determined to be from 5.37×106 to 69 copies of genomic DNA (yellow), 4.40×107 to 1.41×104 copies of pSAM (green), 4.18×108 to 6.69×105 copies of pACYC (pink), and 5.32×108 to 3.41×104 copies of pGEX-6p-1 (blue). Regression analysis displayed R2 values of 0.999 for pSAM, 0.998 for pGEX-6p-1, 0.998 for pACYCDuet, and 0.999 for genomic DNA.
Figure 3.
Plasmid copy number per cell as determined by qPCR.
Plasmid copy numbers were determined in E. coli DH5α harboring one or two vectors. In pink is the copy number of all vectors with the p15A ori; in blue is the copy number of pGEX-6p-1, which is controlled by the pBR322 ori. Plasmids pSAM and pGEX-6p-1 retained their copy numbers both individually and when cells were doubly transformed. This trend for pSAM was similar to that observed with pACYCDuet (a member of the p15A family) both individually and when doubly transformed with pGEX-6p-1. Plasmid pSAMRNAI causes a decrease in the copy number of pGEX-6p-1 when cells were doubly transformed due to the presence of the pET-28a(+)-derived RNAI retained by pSAMRNAI.