boundaries of the origin of replication creation of a pet-28a-derived vector with p15a copy control allowing compatible coexistence with pet vectors边界起源的复制创建一个pet-28a-derived向量与p15a复制控制允许兼容共存与宠物向量.pdfVIP

Boundaries of the Origin of Replication: Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors 1 1,2 Sarmitha Sathiamoorthy , Jumi A. Shin * 1 Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada, 2 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada Abstract 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 1864 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 49 610 and 1464, 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