Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products.
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.
Kim
Han Geun
HG
School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Suwon, Korea.
Kim
Hye Sun
HS
Hwang
Hyun Jin
HJ
Chung
Sung Kyun
SK
Lee
Jung Min
JM
Chung
Dae Kyun
DK
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*
D001427:Bacterial Toxins; D027101:DNA Topoisomerase IV