title: Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products.
abstract: Using linker insertion mutations, we determined the most stable region of the parE gene which encodes a toxic protein (ParE) that inhibits growth of Escherichia coli. The toxicity of ParE was sustained until a 144 bp linker was inserted into this region. We have developed a 3' T-overhang vector based on these characteristics of the GST-ParE toxin, and named pTOC-T. Because pTOC-T uses a post-segregational killing system, all transformants grown up on the plates can be considered as recombinants containing foreign DNA. pTOC-T not require X-Gal, IPTG or other substrates for selection. This T-vector using a positive selection system can be applied to various E. coli strains such as XL1-Blue, BL21, DH5alpha, JM109, and JM110.
authors.affiliation: School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Suwon, Korea.
authors.forename: Han Geun
authors.initials: HG
authors.lastname: Kim
authors.forename: Hye Sun
authors.initials: HS
authors.lastname: Kim
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Hwang
authors.forename: Sung Kyun
authors.initials: SK
authors.lastname: Chung
authors.forename: Jung Min
authors.initials: JM
authors.lastname: Lee
authors.forename: Dae Kyun
authors.initials: DK
authors.lastname: Chung
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D001427:Bacterial Toxins; D027101:DNA Topoisomerase IV