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, 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 does 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.
CITATION STYLE
Kim, H. G., Kim, H. S., Hwang, H. J., Chung, S. K., Lee, J. M., & Chung, D. K. (2004). Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products. Biotechnology Letters, 26(21), 1659–1663. https://doi.org/10.1007/s10529-004-3518-z
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