, Volume 26, Issue 21, pp 1659-1663

Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products

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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, DH5α, JM109, and JM110.

Revisions requested 28 July 2004; Revisions received 7 September 2004