Boundaries of the Origin of Replication: Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors.

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replication (ori); pET-28a(+) contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+) replicon--comprising the ori, RNAI, RNAII, and Rom--with the p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. This essential need is currently not filled. [ABSTRACT FROM AUTHOR]