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Copy-control tightly regulated expression vectors based on pBAC/oriV.
Methods Mol Biol. 2004; 267:155-67.MM

Abstract

A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expression is much more tightly regulated than for the conventional multicopy expression vectors. The simplest version of these copy-control expression vectors is based on the pBAC/oriV plasmid that carries the trfA up-mutant gene under control of the l-arabinose-inducible Para promoter (araC-PBAD). The same promoter controls expression of a gene cloned into MCS. Thus, addition of the inducer (l-arabinose) simultaneously turns on amplification of the plasmid and expression of the cloned gene. Net result is about a 50,000-fold increase in the cloned gene expression. However, when not induced, background expression level is very low, which is important for the maintenance of any "toxic" genes. This vector could be used in most E. coli hosts. Similar versions of the described vector employ the rhamnose-inducible Prha promoter (rhaS-Prha). Other expression systems allow independent regulation of the plasmid amplification and of the cloned gene expression, and some also use the PLtetO-1 promoter. Copy-control expression vector pETcoco, based on the pT7lacO promoter, is commercially available.

Authors+Show Affiliations

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, USA.No affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15269423

Citation

Wild, Jadwiga, and Waclaw Szybalski. "Copy-control Tightly Regulated Expression Vectors Based On PBAC/oriV." Methods in Molecular Biology (Clifton, N.J.), vol. 267, 2004, pp. 155-67.
Wild J, Szybalski W. Copy-control tightly regulated expression vectors based on pBAC/oriV. Methods Mol Biol. 2004;267:155-67.
Wild, J., & Szybalski, W. (2004). Copy-control tightly regulated expression vectors based on pBAC/oriV. Methods in Molecular Biology (Clifton, N.J.), 267, 155-67.
Wild J, Szybalski W. Copy-control Tightly Regulated Expression Vectors Based On PBAC/oriV. Methods Mol Biol. 2004;267:155-67. PubMed PMID: 15269423.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Copy-control tightly regulated expression vectors based on pBAC/oriV. AU - Wild,Jadwiga, AU - Szybalski,Waclaw, PY - 2004/7/23/pubmed PY - 2004/10/8/medline PY - 2004/7/23/entrez SP - 155 EP - 67 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol. Biol. VL - 267 N2 - A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expression is much more tightly regulated than for the conventional multicopy expression vectors. The simplest version of these copy-control expression vectors is based on the pBAC/oriV plasmid that carries the trfA up-mutant gene under control of the l-arabinose-inducible Para promoter (araC-PBAD). The same promoter controls expression of a gene cloned into MCS. Thus, addition of the inducer (l-arabinose) simultaneously turns on amplification of the plasmid and expression of the cloned gene. Net result is about a 50,000-fold increase in the cloned gene expression. However, when not induced, background expression level is very low, which is important for the maintenance of any "toxic" genes. This vector could be used in most E. coli hosts. Similar versions of the described vector employ the rhamnose-inducible Prha promoter (rhaS-Prha). Other expression systems allow independent regulation of the plasmid amplification and of the cloned gene expression, and some also use the PLtetO-1 promoter. Copy-control expression vector pETcoco, based on the pT7lacO promoter, is commercially available. SN - 1064-3745 UR - https://www.unboundmedicine.com/medline/citation/15269423/Copy_control_tightly_regulated_expression_vectors_based_on_pBAC/oriV_ L2 - https://dx.doi.org/10.1385/1-59259-774-2:155 DB - PRIME DP - Unbound Medicine ER -