New shuttle vectors for Escherichia coli and Bacillus subtilis. I. Construction and characterization of plasmid pHY460 with twelve unique cloning sites

Abstract

We have constructed chimeric plasmid vectors, pHY460 and pHY310, from the streptococcal tetracycline resistance (TcR) plasmid pAM alpha 1 (9.2 kb) and the Escherichia coli vector pACYC177 (3.7 kb). These bifunctional plasmids can replicate and express the TcR gene in both E. coli and Bacillus subtilis. Plasmids pHY460 (7.0 kb) and pHY310 (4.8 kb) contain the TcR gene of pAM alpha 1 and the ampicillin resistance (ApR) gene of pACYC177. Both plasmids showed high transformation efficiency in both host cells. pHY460 was maintained stably in B. subtilis and, thus, is a useful shuttle vector functioning in E. coli and B. subtilis. The PvuI, PstI, BglI and BanI sites in the ApR gene and the HpaI, BalI and EcoRV sites in the TcR gene can be used for selection of recombinant plasmids by insertional inactivation. In addition, plasmid pHY460 has unique sites for SacII, BstEII, XbaI, AvaI and BamHI.