An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis.

The recently described beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis [Haima et al., Gene 86 (1990) 63-69]was optimized in several ways. First, the efficiency of translation of the lac Z delta M15 gene was improved. Second, the plasmid-borne lacZ delta M15 gene was segregationally stabilized by integration into the B. subtilis chromosome. Third, a new lacZ alpha complementing cloning vector was constructed, containing more unique target sites. It was shown that large heterologous DNA fragments (up to at least 29 kb) could be cloned with lacZ alpha-complementing vectors carrying the replication functions of the cryptic B. subtilis plasmid pTA1060, and that these inserts were structurally stably maintained for at least 100 generations of growth.