One popular pBR322 derivative is the pET vector series, which contains the T7 promoter-driven system first developed by Studier and Moffatt. The success of these plasmids has been largely due to early characterization of the molecule, including its nucleotide sequence. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.įirst described by Bolivar and Rodriguez, the pBR322 plasmid and its derivatives continue to be among the most widely used cloning vectors in laboratories worldwide (for a review, see ref. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This work was supported by a Collaborative Health Research Project Grant (CHRP 385829, ), National Sciences and Engineering Research Council Discovery Grant (NSERC, ), Canadian Foundation for Innovation/Ontario Innovation Trust New Opportunities Grant (CFI/OIT 7856), and the University of Toronto. Received: JAccepted: SeptemPublished: October 22, 2012Ĭopyright: © Sathiamoorthy, Shin. This essential need is currently not filled.Ĭitation: Sathiamoorthy S, Shin JA (2012) Boundaries of the Origin of Replication: Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. 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. 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☒ in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Swapping of the ori is a common practice however, it is vital that all regions of the original replicon be removed. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49☑0 and 14±4, respectively, when both were present within the same cell. 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. 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. 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. 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.
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