Far different levels of gene expression provided by an oriented cloning system in Bacillus subtilis and Escherichia coli.

A gene expression system for both Bacillus subtilis and Escherichia coli was developed. The expression vector, pHASH102, produces any combination of promoter and open reading frame to be expressed based on the T-extended cloning method. Because the pHASH series vectors are designed to shuttle between the genome and a high copy plasmid in B. subtilis, the expression profiles of copy number dependence can be examined systematically. We demonstrated that vectors with Pr, Pspac, and PS10 promoters are suitable for the overexpression of GFPuv. Moreover, aadK encoding aminoglycoside 6-adenylyltransferase (a streptomycin-resistance gene) of B. subtilis was successfully overexpressed in both B. subtilis and E. coli. These highly expressed GFPuv and aadK genes can be used as a genetic marker for both organisms.